gene regulation & epigenetics concepts and...

1

Gene Regulation amp Epigenetics

Concepts and Applications to Drive Discovery

Dandan Zhong PhD

Senior Field Application Scientist

Merck Millipore Bioscience

2012 December

2

Millex filter

Amicon Ultra

Immobilon PVDF

Millipore

2

About Merck Group

Established in 1668 in Germany

The oldest pharmaceutical and chemical company in the world

40000 employees in 67 country worldwide

Total revenues of EUR 93 billion in 2010

Acquired Millipore in 2010

wwwhistorymerckde

Headquarter

Darmstadt Germany

Merck amp Millipore A Perfect Fithellip

3

Millipore becomes the Life Science Division of Merck in the Chemicals Sector

Chemicals

(Business Sector)

Pharmaceuticals

(Business Sector)

Merck KGaA

Merck Millipore

(Division)

Performance Materials

(Division)

Merck Serono

(Division)

Consumer Health Care

(Division)

6

Star Product lines

bull Epigenetics

bull Drug Screening

bull Stem Cell

bull Neuroscience

bull Metabolic disease

bull （ELISA amp Milliplex）

bull Apoptosis

bull wwwapoptosiscom

now a part of Merck Millipore

4

7

Merck Millipore is a division of

bull Small Molecule Inhibitors

bull Oncology

bull Prokaryotic Expression（PET）

bull Insect Expression

bull Protein Extraction amp Purification


8

Drug Discovery

Cell Signaling

Epigenetics

Stem Cells

Neuroscience

Antibodies

Assay Kits

Instruments

Biotools

Cell culture media amp plates

Cells

Merck Millipore Today Total Solutions for Life Science Research

Cancer Research

Protein Research Inhibitors amp Kinases

5

Epigenetics vs Genetics

Central Dogma

DNA Protein RNA

Classical genetics

6

Typical Example of Epigenetics Regulation

12

7

What is Epigenetics

13

Definition of Epigenetics

Changes gene expression

No Change in DNA sequence

Heritable but potentially reversible

DNA Protein RNA

Epigenetic Potential for Therapeutic Intervention

Most dynamic Most permanent

Deletion

(amplification)

Chromosomal

Translocation

Point mutations

Genetics

Chromatin

Changes

Promoter

Methylation

Silencing

Cell-cycle

Regulated

Changes

Transcription

Factors

Response To

Environmental

Stimuli

Epigenetics

Adapted from James Herman

DNMT1i

Decitabine-Dacogen 2006

HDACi Vorinostat-Zolinza 2006

8

16

How to Study Epigenetics

Agenda

Concepts in Epigenetics

Chromatin

Histone Modifications

Gene regulation by RNAs

DNA Methylation

Epigenetics Tools and Applications

Critical considerations and experimental design

(experimental successes and potential pitfalls)

ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

9

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

Chromatin Packaging

Eukaryotic DNA is packaged as chromatin

Histone octamer

ndash 2 molecules each of H2A H2B H3 amp H4

Histone Octamer + DNA

ndash Nucleosome

H1 + Nucleosome

ndash 30 nm fiber

ndash Scaffolding Proteins (Nuclear Matrix Attachment)

ndash Topoisomerases (Supercoiling)

Multiple levels of compaction

Heterochromatin vs Euchromatin

Felsenfeld et al Nature 2003

10

20

Histones

H1 H2A H2B H3 H4

bullRich in Arg amp Lys (Stablize the electrostatic

interaction with DNA)

bullMost conserved proteins

The Universe of Histone Modifications

Regulate biological function either by

bull Recruiting other protein ldquoreaders writers erasersrdquo

bull Changing chemical structure to allow DNA access or nucleosome

repositioning or remodeling

bull ldquoHistone coderdquo

Bhaumik et al NSMB 2007

11

22

Histone Covalent Modifications

bull Both N- and C- terminals

bull Most Modifications on N-terminal

Acetylation

Phosphorylation

Methylation

Ubiquitination

Sumoylation

Citrullination

Ribosylation

Crotonylation

Biotinylation


23

Acetylation

bull Histone aceytltransferase (HAT)

bull Histone deacetylase (HDAC)

bull Reversible amp Dynamic

bull Acetylation activates gene transcription

bull Deacetylation acts contrarily

P300

PCAF H2B

PEPAKSAPAPKKGSKKAVTKAQKK 5 12 15 20

Ac Ac Ac Ac

SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 5 8 12 16

H4

Ac H2A

SGRGKQGGKARAKAKSRSSRAGLQ 5 9

Ac

H3

ARKSTGGKAPRKQLATKAARKSAP 9 14 18 23 27

Ac Ac Ac Ac Ac

Ac Ac Ac Ac

12

24

HATsHDACs amp CancerCancer therapy

o Many tumor suppressor genes are silenced by a mechanism involving DNA methylation and histone

deacetylation contributing to many types of cancer HDAC inhibitors currently show tremendous early promise as

treatment agents

o The 1st drug of this type suberoylanilide hydroxamic acid (Verinostat MSD) was approved by the FDA for the

treatment of cutaneous T-cell lymphoma in 2006

33

Methylation

JHDM3 LSD1 PAD4

JHDM3

bull On Arg Lys

bull Reversible

bull Single- di- tri- methylation

bull Activate or repress gene expression

Regulate

bull Cell cycle

bull Cell proliferation

bull Chromatin structure

bull Early development

13

Disease Associated Genetic Alterations in Protein Methyltransferases

Copeland Drug Discovery

Today In Press 2011

36

Phosphorylation

Main function

bullCell cycle

bullMeiosis

bullApoptosis

bullDNA damage repair

bullDevelopment

bullCell division

H2A

SGRGKQGGKARAKAKSRSSRAGLQ

P

1

H2B

PEPAKSAPAPKKGSKKAVTKAQKK 14

P

H3

ARTKQTARKSTGGKAPRKQLATKAARKSAP 3 1011 28

P P P P

SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 1 18

H4 P P

bull On Ser Thr

14

38

Histone H2AX Phosphorylation Ser139

H2AX variant of H2A has unique C-term

sensor for DNA double-strand breakage

involve in DNA damage repair

H2AX deficiency related to tumorigenesis

bullGood marker for early apoptosis

bullSensitive readout for DNA damage

H2AX Phosphorylation assay (Cat 17-327)

Cell-based ELISA （CELISA）

Log phase NIH3T3A31 cells were treated for 2

hours with increasing Etoposide

40

H2AX Phosphorylation Assay Kit （17-327）

bull Chemiluminescent

bull Detect signals in cells

bull Avoiding lysing cells to protect Phosphorylation signal

15

42

H2B Serine 14 Phosphorylation Apoptosis marker

Detection of H2B phosphorylated at serine 14 in

HL-60 cells stimulated to undergo apoptosis by

treatment with VP-16 Left anti-phospho

histone H2B (Ser14) (07-191) staining Right

DAPI staining Courtesy Dr David Allis The

Rockefeller University

Asynchronous HeLa cells stained with anti-

phospho H3 (Ser10 ) (cat 06-570) in red

and anti-beta tubulin (cat 05-661) in green

Serine 10 phosphorylation of H3 is almost

exclusively observed on condensing mitotic chromosomes (arrows)

Phosphorylation of H3 ( Ser10)

cell mitosis marker

Left anti-phospho

histone H2B (Ser14)

(07-191)

Right DAPI

44

Ubiquitinization

Single Ubiquitin ligation（different from Hydrolysis）

H2A(5-15)和H2B(1-15)

Change Chromatin structure expose DNA start transcription

Recruit transcription factor

Regulation other Histone modifications

eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation


H2A

119

u

120

H2B

PEPAKSAPAPKKGSKKAVTKAQKK

u

16

45

Sumoylation

Small ubiquitin-related modifier (SUMO)

Not hydrolysis

H2AH2BH4

Inhibit gene transcription

Merck Millipore Provide

bull anti-SUMO-1 anti-SUMO-23 抗体

bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme

46

Citrullination

bull Rheumatoid athritis

bull MS

bull Alzheimerrsquos disease

17

Histone Code Where To Start

H3 N- hellipK4hellipK9S10helliphellipK27hellipK36

me2 me3

Ac

me3

Ac

Ph

Ac

me3

- active promoters -repressed genes (PRC2)

bivalent promoters

- transcribed exons - heterochromatin

me3

me1 - active enhancers

me3 - bivalent promoters

- active genes

- active genes - active enhancers

- mitosis

Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome

Example of the actively transcribed gene

Bell et al Nature Reviews Genetics 2011

18

High Low Probability

Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator

Ernst et al Nature Biotechnology 2010

Genomic Elements and Chromatin State

TSS

UTR

Exons

Intron Promoter Enhancer

LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin


Chromatin Associated Non-Coding RNAs

DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA

Why we need ChIP analysis

Transcriptional Regulation

Transcription factors chromatin modifiershellip

ChIP is the most common in vivo method used to study

endogenous proteins associated with chromatin

ChIP can show the actual gene regulation under physiological

status

Chromatin IP (ChIP)

A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins

56

ChIP Workflow

N - + I

Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation

+- crosslinking harvest and count cells

Fragmentation Sonication enzymatic treatment

Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG

agarose or magnetic beads

DNA Recovery Digest protein remove beads

reverse crosslinks extractpurify DNA

Detection PCR qPCR Microarray

1

2

3

4

5

7

6

20

Critical Considerations for Successful ChIP

bull Experimental design

ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)

bull Chromatin preparation

ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent

bull Antibody quality and IP strategy

ndash Use well characterized antibodies

ndash Consider all available applications for unknowns

bull Data Analysis

ndash Utilize controls consider assay performance (qPCR standard curve)

ndash Test enrichment by qPCR before running genome wide experiment

WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59

Experimental Designcell number

Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter

Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate

High Total Histone H3 1 X 105 100

Medium-High Acetyl Histone H3 5 X 105 20

Medium RNA Polymerase II 1 X 106 10

Low Transcription Factor X 2 X 107 05

Extremely Low Liver specific Txn Factor infin NA

1

21

Standard ChIP Experiment with Biological Validation

ChIP Ab+ Antibodyprimer set (Cat 17-622)

specifically detects Trimethyl-Histone H3

(Lys27) with human α-Satellite (Fold

Enrichment relative to mock IgG control)


specifically detects Trimethyl-Histone H3 (Lys27)

with human Hela cells (Fold increase relative to

mock IgG control)

Mock IP vs Specific IP is useful

but location controls are valuable toohellip

Experimental Design 1

Mock IP vs Specific IP is useful but location controls are valuable toohellip

002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter

17-10109 ChIPAb+ Tcf-4

22

Fixation

Does not require fixation

ndash Gentler but less robust method

ndash May require more cells

ndash Often used with enzymatic digestion instead

of sonication

ndash Formaldehyde crosslinking may mask epitope

Requires high affinity interactions

ndash Histones and Histone modifications

ndash Requires fixation (ie

Formaldehyde 1 10min at

37degC)

ndash More common for detecting

weaker interactions

ndash Interrogation of complexes

ndash Over-crosslinking can

increase artifactual

interactions

ndash Crosslinking can mask the

epitope

Native ChIP (N-ChIP) ChIP (X-ChIP)

2

Chromatin Fragmentation

Required to solubilize chromatin

Produce fragments from 300 bp to 1 Kb

Largely random fragmentation

Many instrument models with varying probe sizes

power settings hellip

Performance varies with volume of sample and

cell density

Foaming denaturation shearing of target protein

Can be challenging requires optimization

Sonication

bull Micrococcal nuclease

bull Can achieve single nucleosome resolution

bull Restriction endonucleases

Enzymatic Treatment

Nucleosomal

Ladder

Increasing Cycles of Sonication

Cat 17-375 EZ-Zyme

Cat 12-704 ChIPable Chromatin HEK293

Cat 12-705 ChIPable Chromatin HT29

Cat 12-687 ChIPable Chromatin HeLa

ChIPable Chromatin Sonicated formalhehyde-fixed

chromatin

TroubleShooting

Has the DNA shearing been

checked on an agarose gel

3

23

69

Antibody

Not all antibodies are suitable for ChIP

Specificity of epitope is critical

bull Epitope(s) must be exposed in native conformation

Epitope(s) must not be masked by crosslinking

Antibody Selection Polyclonal vs Monoclonal Purified vs Crude

Use ChIP validated Abs where possible

If unavailable consider IHC EMSA IP validated antibodies

ChIP validated Abs

4

70

IP Magnetic vs Agarose Beads

Agarose Beads

More cost effective

High background need blocking

(BSA carrier DNA)

Pre-clear samples

Perform -+ antibody controls

Magnetic Beads

No centrifugation Fast

Smooth surface No need blocking

Less unspecific binding

Less Sample amp less Ab

Ability to automate Ideal for high

throughput

ChIP on chip where salmojn sperm DNA

can be problematic

5

24

DNA Recovery And Detection 6

Safety and disposal issues

Perceived as cost effective

Useful for low amount of DNA

Easily have protein contaminant

Improved Purity

Easier to handle

Faster processing time

PhenolChloroform Column Purification

In the classical ChIP Kit In the EZ-ChIP Kit

72

Detection method after ChIP

PCRqPCRGene specific known binding sequence

ChIP-CloningFind unknown binding sequence

ChIP-SequencingFind unknown binding sequence

ChIP reChIPDetect the co-binding sequence by two

proteins

Genomic tiling micro-array (ChIP chip)Genome-wide

analysis

7

25

Concerns of Researchers doing ChIP

Successful ChIP

Lower background

Specificity

Repeatable results

Presentation title in footer | 00 Month 0000 73

An Assessment of Histone-Modification Antibody Quality by modENCODE Group

Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies

26

Antibody Quality--Validation Data

H3K4me3 affinity purified

Polyclonal Ab Cat 07-473

No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking

H3K4me3 Recombinant Rabbit

Monoclonal Ab Cat 05-745R

H3K4me3 H3K4me3gtH3K4me2

40 ng

4 ng

Dot Blot

All peptides contain H3K4me23

Peptide Microarray

Biological Specificity in ChIP

AbSurancetrade Histone Antibody Specificity Arrays

Cat 16-665 Histone H2A H2B H4 Array

Cat 16-667 Histone H3 Array

Cat 16-668 Complete set (H2A H2B H3 H4 Array)

AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity

Reproducible results


79

How to get reproducible ChIP results

Accurate Cell counts

超声周期的增加酶消化

28

bull Speedy 14sec

bull Accurate

Cell Count Accuracy mdashmdash the basis for good and reproducible results

82




Same assay condition


bull Sonication optomization same

sonication condition for same

sample amount and sample

volume

bull Enzymn digestion

bull DNA input

29

83

Merck Millipore provides 3 Generation of ChIP kits

17-295 ChIP Kit

17-245 ChIP Acetyl H3


17-371 EZ-ChIP

17-375 EZ-Zyme

ChIP (bare bonesmdashexperienced users)

EZ-ChIP (controls buffers spin filtersmdashNew users)

Magna ChIP (faster cleaner higher throughput)

17-61011 Magna ChIP Protein AG (buffers spin filters)

17-4089 EZ-Magna ChIP AG (complete controls)

1

2

3

The first ChIP kit in the world

Most citation

Magna ChIPtrade G Tissue Kit


New

300 microm of coronal mouse brain section with region specific

microdissection of the hippocampus (A) and cerebral cortex

(B) The isolated tissue was placed above the dissected region

(a hippocampus b cortex)

bullOptimized protocol for Tissue ChIP

Reproducible amp reliable ChIP from

small amounts of tissue

Site specific ChIP analysis using

Microdissection of 1mm3 tissue

sections using included tool

Includes tissue stabilization and

lysis reagents

Proven performance in a variety of

tissues

Expert support from the leader in

chromatin immunoprecipitation

bullTissue samples are more complex and the cells are

heterogeneous thus making ChIP analysis less amenable

30

Magna ChIPtrade HT96 17-10077 17-10078

85

17-10071 Magna GrIP HY96 Rack

New

bull High throughput

bull Less sample

- 104~105 cellswell

bull Less Antibody

bull Better reproducibility

bull Easier to perform

- Single buffer system

- Protein AG blend

86

ChIP Ab+

bull Include negative control Ab

bull qPCR control primers

bull Strict QC Every lot gets ChIP and qPCR tested

Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs

bullgt49 Histone-Modifying Enzyme Abs

Donrsquot forget high quality ChIPable Abs

gt20000 Epigenetic papers using MM antibodies

31

ChIP Related Products and Accessories

EZ-Zyme Chromatin Prep Kit (cat 17-375)

Enzymatic approach

Alternative to sonication

Compatible with cross-linked or native chromatin

Magna ChIP Protein A Beads (cat16-661 and 16-661X)

Magnetic beads optimized for ChIP

Magna ChIP Protein G Beads (cat 16-662 and 16-662X)


Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)


Magna GrIP 8-well Rack (Cat 20-400 )

For Magna ChIP beadskits only Low cost option

PureProteometrade Magnetic Stand (Cat LSKMAGS08)

Universal Rack for protein IP or ChIP

Feature rich rack

Protein A Agarose Beads (Cat 16-157)

Blocked for ChIP

Protein G Agarose Beads

Cat 16-201 blocked for ChIP

EZ-Zyme Digested Chromatin

Nucleosomal

Ladder

90

Histone amp Histone modifying Enzyme Products

Antibodies166 Histone and Modified Histone Abs

49 Histone-Modifying Enzyme Abs

Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT

Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)

Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-

Histone H3 (Ser10) Assay (HCS204) etc

Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)

Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying

Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control

Histone Protein and Peptides

InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium

Butyrate (19-137) 等

32

Agenda


Chromatin


Gene Regulation by RNAs

DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92

What Happens After Transcription Post Transcriptionhellip

ChIPTranscriptional Regulation


Post-transcription regulation

has pivotal roles for development metabolism neuronal function and aging

Splicing transport localization translation degradation of mRNA and non-

coding RNAs

temporally and coordinately influence the rate of protein synthesis

33

96

Various function of RNA

RNA Function Messenger RNAs

(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)

Translation (sequence recognition)

Transfer RNAs

(tRNA)

Translation (amino-acyl peptide formation)

7S xist piwi others Specialized Functions

(Telomeres X inactivation)

Non-coding RNAs

(ncRNA miRNAmicroRNA siRNA shRNA)

RNA Stability and Translation

RNA-binding proteins (RBPs)

bull Play a key role in post-transcriptional regulation of expression

bull Involved in every step of mRNA and ncRNA metabolism

bull mRNA Processing (capping polyadenylation)

bull RNA splicing

bull Stability

bull Transport

bull Translational regulation (rate amount of protein synthesis

bull ncRNA processing and function

34

98

~10 or more of disease associated

point mutations affect splicing

Examples of Diseases involving RBPs and RNA

Disease Genemutation Function

Fragile X Mental Retardation FMRP mRNA Transport

Spinal Muscular Atrophy (SMA) SMNs Splicing

Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators

Cancer miR-372 miR-373 RNA interference

Cancer miR-17-92 cluster RNA interference

Cancer SFRS1 Splicing translation export

Cancer RBM5 Splicing

RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way

RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP

Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs

The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)

35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102

Merck Millipore provides RIP kits amp RIPAb+

03-101 RIPAb+ PABPC1

03-102 RIPAb+ HuR

03-103 RIPAb+ SNRNP70

03-104 RIPAb+ CUGBP1

03-105 RIPAb+ Lin28

03- 248 RIPAb+ pan Ago

03- 246 RIPAb+ FXR2

03- 245 RIPAb+ Hexim 2

03- 205 RIPAb+ hnRNP C1C2

03- 206 RIPAb+ hnRNP U

03- 116 RIPAb+ STAU1(Staufen1)

03- 107 RIPAb+ EF1alpha

amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt

B-Actin RT-PCR HeLa RIP IP

Part Product Reaction

17-701 EZ-Magna RIP Kit 12

(includes positive controls)

17-700 Magna RIP Kit 12

17-704 Magna RIP Quad Kit 48

36

103

RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip

04-321 Anti-4E-BP1 Rabbit Monoclonal

05-490 Anti-ACTRAIB1 clone AX15

07-599 Anti-Ago 1

07-590 Anti-Ago 2

04-085 Anti-Ago family

04-083 Anti-Ago1 clone 6D82

04-642 Anti-Ago2 clone 9E82

04-084 anti-Ago2 clone 9E82

05-967 Anti-Ago4 clone 5F92

09-456 Anti-AIRE 100 microg

07-210 Anti-AKAP 150 200 microL


06-417 Anti-AKAP 95 200 microg

07-260 Anti-AUF1

AB10004 anti-BARD1 100 microL

AB16507 Anti-Bnip3L internal 100 microg

05-641 Anti-BRAF35 clone 421

07-306 Anti-CtBP-1 200 microg

05-621 Anti-CUG-BP1 clone 3B1 200 microg

04-047 Anti-CuGBP2 Clone 1H2 100 microg

04-721 Anti-Dicer1 clone 5D122

07-717 Anti-Drosha

07-397 Anti-Ebp1

05-235 Anti-EF1α clone CBP-KK1

07-589 Anti-EF2 Kinase

AB3251 Anti-eIF4E Binding Protein

04-347 Anti-eIF-4E CT Rabbit Monoclonal

07-1568 Anti-ESETSetDB1


MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL

07-504 Anti-hnRNP A0

04-088 Anti-hnRNP K clone F45P9C7

05-620 Anti-hnRNP M1-M4 clone 1D8

05-689 Anti-HP1α clone1519s2 200 microg

AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR

AB15506 Anti-Iron regulatory protein 1 50 microg

AB15508 Anti-Iron Regulatory Protein 2

MAB5582 Anti-Iron Regulatory Protein 2

07-1385 Anti-Lin28

04-048 Anti-MBNL clone 3A4

07-575 Anti-MDM2

MAB3776 Anti-MDM2 clone SMP14

MAB10085 Anti-MSI2 clone 1F2

AB5977 Anti-Musashi

AB15648 Anti-Musashi

07-637 Anti-Nova-1

MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL

05-565 Anti-Nucleolin

AB15244 Anti-NXF2

05-257 Anti-p300 CT clone RW128 200 microg

05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg

05-850 Anti-p68 clone PAb204 200 microg

05-847 Anti-PABP clone 10E10

MAB11015 Anti-PABPC4 clone 6E12

AB3242 Anti-PGC-1

07-823 Anti-phospho eIF4E (Ser209)

07-822 Anti-phospho-eIF2Bε (Ser539)

07-760 Anti-phospho-eIF-2α (Ser51)

04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal

07-824 Anti-phospho-eIF4G (Ser1108)

07-566 Anti-phospho-hnRNP A0 (Ser84)

MAB10104 Anti-PUM2 clone 1E10

AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7

MAB10105 Anti-RBMS3 clone 1H6 100 microL

05-839 Anti-RNase L clone 2G5

AB4146 Anti-RO52 100 microg

MAB385 Anti-S100L Protein clone S100-14 100 microg

06-891 Anti-SMRTe 200 microg

AB5781 Anti-Staufen

MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL

104

The first RIP kit user in China published RIP result on Nature Immunology

37

RNA Detection in Live Cells

SmartFlareTM RNA detection Probe


Novel

bull Simple overnight mix with cells

bull Stable 1-3 days

bull Non-toxic Cells reusable

bull Sensitive

Now you can know Whatrsquos Really Happening


Applications

bull RNA detection in live cells

bull miRNA or mRNA quantification

bull Real time analysis

bull Detection of different biomolecules in

the same sample (ie protein + RNA)

bull Cell Sorting based on an RNA target

bull Isolation of pure cell populations

bull Monitoring of transfection efficiencies

bull Multiplex up to 3 RNA targets

38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)

bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes

bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)

bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated

bull m5C accounts for ~1-8 of total DNA bases

Mammalian DNA methylation patterns vary in time and space

DNA Methylation Modifications

bull Cellular Differentiation stem cell neuronal hydroxymethylation

Methylation Oxidation

39

Functions of DNA Methylation and Connection to Chromatin Remodeling

bullImprinting

bullX-Chromosome Inactivation

bullAging

bullNutrient Sensing

bullCellular Differentiation

bullGenomic Diversity (Transposons)

Feinberg amp Tycko Nature Reviews Cancer 2004

DNA Methylation Technologies

How is it studied

Bisulfite modification followed by MSP or sequencing

Methylated DNA enrichment

ndash Methylated DNA immunoprecipitation (MeDIP)

ndash Enrichment using methylated DNA binding proteins (MBD)

Methylation-specific restriction digestion

ndash Methylation-sensitive (cuts only unmethylated DNA)

ndash Methylation-specific (cuts only methylated DNA)

Many Epigenetics researchers use bisulfite modification as the

technique of choice to study DNA methylation because of high

resolution

Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic

epigenetic modifications

40

A Toolbox for Cytosine Methylation Analysis

Method Advantages Disadvantages

Sodium

Bisulfite

Conversion

Resolution at the nucleotide level bull Harsh chemical treatment of DNA can

cause its damage

bull Potential incomplete conversion or

over-conversion artifacts

bull Not 5-mC and 5-hmC specific

MeDIP bull Fast

bull Applicable for NGS and arrays

bull Requires specific antibody

bull May require more than on mC for Ab

binding

bull Requires DNA denaturation

bull Medium level resolution

Methylated

DNA-binding

Proteins

bull No DNA denaturation required



Methylation

Specific

Restriction

Enzymes

bull Easy to use

bull Availability of recombinant

enzymes

bull Limited by the enzyme recognition site

(low resolution)

bull Lower throughput

Platform Technologies for DNA Methylation

Laird Nat Rev Gen 2010

Applications by

Detection

Platform

Resolution amp Sample

Quantity by

Appplication

41

113

MSP

BLAH U U U U U

Challenge 1 Bisulfite Conversion is a Balancing Act

Schumaker methologixcom

Genereux et al NAR 2008

Original ATCGATCATCGATCCAT

Converted ATCGATTATCGATTTAT

Incomplete conversion ATCGATCATCGATTTAT

Inappropriate conversion ATCGATTATTGATTTAT

mC hmC

42

CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple

ndash The fastest most efficient and easiest bisulfite

modification kit from the epigenetics market leader

bull Features and Advantages

ndash Complete protocol in lt90 minutes

ndash 999 conversion efficiency without over-conversion

ndash Convert from 1 ng to 1 mg of DNA

ndash Recover modified DNA in as little as 25 ml

ndash Proven performance in multiple downstream

applications

0

2

4

6

8

Ho

urs

Protocol length 15 3 6

Millipore Supplier Z Supplier Q

18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385

Millipore Supplier Z

DNA Quality Influences The Extent of Bisulfite Conversion

Warnecke et al Methods 2002

Complete protein removal is essential for avoiding artifacts

Non-CpG methylation

observed in crude genomic

preparations in GSTPI promoter

43

119

httpwwwurogeneorgmethprimerindex1html

MSP primer design

Challenge 2 MSP Prime Design

Common methods of DNA methylation

Methylation-Specific PCR(MSP)

DNA Microarray

Restriction Landmark Genomic Scanning

Whole Genome Bisulfite Sequencing

Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing

Bisulfite Modification

CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128

Lastest Epigenetics Brochure

45

Online Tools For Epigenetics Research

To Learn More Please Visit Us Online or contact your local

Representative

wwwmilliporecomepigenetics

130

Chromatin Immunoprecipitation (ChIP)

Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)

Magna ChIPtrade Beads (Protein A G AG)

RNA Immunoprecipitation

Magna RIPtrade (Protein AGwith and without controls)

Bisulfite Modification and Analysis

CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)

CpG Wizreg Methylation Specific PCR Kit

CpGenometrade Methylated and Unmethylated DNA controls

Isolation and Enrichment of Methylated DNA

CpG MethylQuesttrade Methylated DNA isolation kit

MeDIP antibodies against 5meC 5hmeC

Beta-Glucosyltransferase

Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay

HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays

Recombinant Histones dimers tetramers and octamers

Epigenetics Antibodies

ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets

gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors

Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)

EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery

46

131

Questions

Merck Millipore

Unleash the Potential of Life Science

132

asiatechservmerckgroupcom

2

About Merck Group

Established in 1668 in Germany

The oldest pharmaceutical and chemical company in the world

40000 employees in 67 country worldwide

Total revenues of EUR 93 billion in 2010

Acquired Millipore in 2010

wwwhistorymerckde

Headquarter

Darmstadt Germany

Merck amp Millipore A Perfect Fithellip

3


Chemicals

(Business Sector)

Pharmaceuticals

(Business Sector)

Merck KGaA

Merck Millipore

(Division)


(Division)

Merck Serono

(Division)


(Division)

6

Star Product lines

bull Epigenetics

bull Drug Screening

bull Stem Cell

bull Neuroscience



bull Apoptosis



4

7



bull Oncology





8

Drug Discovery

Cell Signaling

Epigenetics

Stem Cells

Neuroscience

Antibodies

Assay Kits

Instruments

Biotools


Cells


Cancer Research


5


Central Dogma

DNA Protein RNA

Classical genetics

6


12

7

What is Epigenetics

13





DNA Protein RNA



Deletion

(amplification)

Chromosomal

Translocation

Point mutations

Genetics

Chromatin

Changes

Promoter

Methylation

Silencing

Cell-cycle

Regulated

Changes

Transcription

Factors

Response To

Environmental

Stimuli

Epigenetics


DNMT1i



8

16


Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

9

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

Chromatin Packaging


Histone octamer



ndash Nucleosome

H1 + Nucleosome

ndash 30 nm fiber






10

20

Histones

H1 H2A H2B H3 H4











11

22




Acetylation

Phosphorylation

Methylation

Ubiquitination

Sumoylation

Citrullination

Ribosylation

Crotonylation

Biotinylation


23

Acetylation






P300

PCAF H2B


Ac Ac Ac Ac


H4

Ac H2A


Ac

H3


Ac Ac Ac Ac Ac

Ac Ac Ac Ac

12

24




treatment agents



33

Methylation

JHDM3 LSD1 PAD4

JHDM3

bull On Arg Lys

bull Reversible



Regulate

bull Cell cycle




13



Today In Press 2011

36

Phosphorylation

Main function

bullCell cycle

bullMeiosis

bullApoptosis


bullDevelopment

bullCell division

H2A


P

1

H2B


P

H3


P P P P


H4 P P

bull On Ser Thr

14

38












40





15

42














cell mitosis marker

Left anti-phospho

histone H2B (Ser14)

(07-191)

Right DAPI

44

Ubiquitinization


H2A(5-15)和H2B(1-15)






H2A

119

u

120

H2B


u

16

45

Sumoylation


Not hydrolysis

H2AH2BH4





46

Citrullination


bull MS


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


3


Chemicals

(Business Sector)

Pharmaceuticals

(Business Sector)

Merck KGaA

Merck Millipore

(Division)


(Division)

Merck Serono

(Division)


(Division)

6

Star Product lines

bull Epigenetics

bull Drug Screening

bull Stem Cell

bull Neuroscience



bull Apoptosis



4

7



bull Oncology





8

Drug Discovery

Cell Signaling

Epigenetics

Stem Cells

Neuroscience

Antibodies

Assay Kits

Instruments

Biotools


Cells


Cancer Research


5


Central Dogma

DNA Protein RNA

Classical genetics

6


12

7

What is Epigenetics

13





DNA Protein RNA



Deletion

(amplification)

Chromosomal

Translocation

Point mutations

Genetics

Chromatin

Changes

Promoter

Methylation

Silencing

Cell-cycle

Regulated

Changes

Transcription

Factors

Response To

Environmental

Stimuli

Epigenetics


DNMT1i



8

16


Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

9

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

Chromatin Packaging


Histone octamer



ndash Nucleosome

H1 + Nucleosome

ndash 30 nm fiber






10

20

Histones

H1 H2A H2B H3 H4











11

22




Acetylation

Phosphorylation

Methylation

Ubiquitination

Sumoylation

Citrullination

Ribosylation

Crotonylation

Biotinylation


23

Acetylation






P300

PCAF H2B


Ac Ac Ac Ac


H4

Ac H2A


Ac

H3


Ac Ac Ac Ac Ac

Ac Ac Ac Ac

12

24




treatment agents



33

Methylation

JHDM3 LSD1 PAD4

JHDM3

bull On Arg Lys

bull Reversible



Regulate

bull Cell cycle




13



Today In Press 2011

36

Phosphorylation

Main function

bullCell cycle

bullMeiosis

bullApoptosis


bullDevelopment

bullCell division

H2A


P

1

H2B


P

H3


P P P P


H4 P P

bull On Ser Thr

14

38












40





15

42














cell mitosis marker

Left anti-phospho

histone H2B (Ser14)

(07-191)

Right DAPI

44

Ubiquitinization


H2A(5-15)和H2B(1-15)






H2A

119

u

120

H2B


u

16

45

Sumoylation


Not hydrolysis

H2AH2BH4





46

Citrullination


bull MS


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


4

7



bull Oncology





8

Drug Discovery

Cell Signaling

Epigenetics

Stem Cells

Neuroscience

Antibodies

Assay Kits

Instruments

Biotools


Cells


Cancer Research


5


Central Dogma

DNA Protein RNA

Classical genetics

6


12

7

What is Epigenetics

13





DNA Protein RNA



Deletion

(amplification)

Chromosomal

Translocation

Point mutations

Genetics

Chromatin

Changes

Promoter

Methylation

Silencing

Cell-cycle

Regulated

Changes

Transcription

Factors

Response To

Environmental

Stimuli

Epigenetics


DNMT1i



8

16


Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

9

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

Chromatin Packaging


Histone octamer



ndash Nucleosome

H1 + Nucleosome

ndash 30 nm fiber






10

20

Histones

H1 H2A H2B H3 H4











11

22




Acetylation

Phosphorylation

Methylation

Ubiquitination

Sumoylation

Citrullination

Ribosylation

Crotonylation

Biotinylation


23

Acetylation






P300

PCAF H2B


Ac Ac Ac Ac


H4

Ac H2A


Ac

H3


Ac Ac Ac Ac Ac

Ac Ac Ac Ac

12

24




treatment agents



33

Methylation

JHDM3 LSD1 PAD4

JHDM3

bull On Arg Lys

bull Reversible



Regulate

bull Cell cycle




13



Today In Press 2011

36

Phosphorylation

Main function

bullCell cycle

bullMeiosis

bullApoptosis


bullDevelopment

bullCell division

H2A


P

1

H2B


P

H3


P P P P


H4 P P

bull On Ser Thr

14

38












40





15

42














cell mitosis marker

Left anti-phospho

histone H2B (Ser14)

(07-191)

Right DAPI

44

Ubiquitinization


H2A(5-15)和H2B(1-15)






H2A

119

u

120

H2B


u

16

45

Sumoylation


Not hydrolysis

H2AH2BH4





46

Citrullination


bull MS


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


5


Central Dogma

DNA Protein RNA

Classical genetics

6


12

7

What is Epigenetics

13





DNA Protein RNA



Deletion

(amplification)

Chromosomal

Translocation

Point mutations

Genetics

Chromatin

Changes

Promoter

Methylation

Silencing

Cell-cycle

Regulated

Changes

Transcription

Factors

Response To

Environmental

Stimuli

Epigenetics


DNMT1i



8

16


Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

9

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

Chromatin Packaging


Histone octamer



ndash Nucleosome

H1 + Nucleosome

ndash 30 nm fiber






10

20

Histones

H1 H2A H2B H3 H4











11

22




Acetylation

Phosphorylation

Methylation

Ubiquitination

Sumoylation

Citrullination

Ribosylation

Crotonylation

Biotinylation


23

Acetylation






P300

PCAF H2B


Ac Ac Ac Ac


H4

Ac H2A


Ac

H3


Ac Ac Ac Ac Ac

Ac Ac Ac Ac

12

24




treatment agents



33

Methylation

JHDM3 LSD1 PAD4

JHDM3

bull On Arg Lys

bull Reversible



Regulate

bull Cell cycle




13



Today In Press 2011

36

Phosphorylation

Main function

bullCell cycle

bullMeiosis

bullApoptosis


bullDevelopment

bullCell division

H2A


P

1

H2B


P

H3


P P P P


H4 P P

bull On Ser Thr

14

38












40





15

42














cell mitosis marker

Left anti-phospho

histone H2B (Ser14)

(07-191)

Right DAPI

44

Ubiquitinization


H2A(5-15)和H2B(1-15)






H2A

119

u

120

H2B


u

16

45

Sumoylation


Not hydrolysis

H2AH2BH4





46

Citrullination


bull MS


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


6


12

7

What is Epigenetics

13





DNA Protein RNA



Deletion

(amplification)

Chromosomal

Translocation

Point mutations

Genetics

Chromatin

Changes

Promoter

Methylation

Silencing

Cell-cycle

Regulated

Changes

Transcription

Factors

Response To

Environmental

Stimuli

Epigenetics


DNMT1i



8

16


Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

9

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

Chromatin Packaging


Histone octamer



ndash Nucleosome

H1 + Nucleosome

ndash 30 nm fiber






10

20

Histones

H1 H2A H2B H3 H4











11

22




Acetylation

Phosphorylation

Methylation

Ubiquitination

Sumoylation

Citrullination

Ribosylation

Crotonylation

Biotinylation


23

Acetylation






P300

PCAF H2B


Ac Ac Ac Ac


H4

Ac H2A


Ac

H3


Ac Ac Ac Ac Ac

Ac Ac Ac Ac

12

24




treatment agents



33

Methylation

JHDM3 LSD1 PAD4

JHDM3

bull On Arg Lys

bull Reversible



Regulate

bull Cell cycle




13



Today In Press 2011

36

Phosphorylation

Main function

bullCell cycle

bullMeiosis

bullApoptosis


bullDevelopment

bullCell division

H2A


P

1

H2B


P

H3


P P P P


H4 P P

bull On Ser Thr

14

38












40





15

42














cell mitosis marker

Left anti-phospho

histone H2B (Ser14)

(07-191)

Right DAPI

44

Ubiquitinization


H2A(5-15)和H2B(1-15)






H2A

119

u

120

H2B


u

16

45

Sumoylation


Not hydrolysis

H2AH2BH4





46

Citrullination


bull MS


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


7

What is Epigenetics

13





DNA Protein RNA



Deletion

(amplification)

Chromosomal

Translocation

Point mutations

Genetics

Chromatin

Changes

Promoter

Methylation

Silencing

Cell-cycle

Regulated

Changes

Transcription

Factors

Response To

Environmental

Stimuli

Epigenetics


DNMT1i



8

16


Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

9

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

Chromatin Packaging


Histone octamer



ndash Nucleosome

H1 + Nucleosome

ndash 30 nm fiber






10

20

Histones

H1 H2A H2B H3 H4











11

22




Acetylation

Phosphorylation

Methylation

Ubiquitination

Sumoylation

Citrullination

Ribosylation

Crotonylation

Biotinylation


23

Acetylation






P300

PCAF H2B


Ac Ac Ac Ac


H4

Ac H2A


Ac

H3


Ac Ac Ac Ac Ac

Ac Ac Ac Ac

12

24




treatment agents



33

Methylation

JHDM3 LSD1 PAD4

JHDM3

bull On Arg Lys

bull Reversible



Regulate

bull Cell cycle




13



Today In Press 2011

36

Phosphorylation

Main function

bullCell cycle

bullMeiosis

bullApoptosis


bullDevelopment

bullCell division

H2A


P

1

H2B


P

H3


P P P P


H4 P P

bull On Ser Thr

14

38












40





15

42














cell mitosis marker

Left anti-phospho

histone H2B (Ser14)

(07-191)

Right DAPI

44

Ubiquitinization


H2A(5-15)和H2B(1-15)






H2A

119

u

120

H2B


u

16

45

Sumoylation


Not hydrolysis

H2AH2BH4





46

Citrullination


bull MS


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


8

16


Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

9

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

Chromatin Packaging


Histone octamer



ndash Nucleosome

H1 + Nucleosome

ndash 30 nm fiber






10

20

Histones

H1 H2A H2B H3 H4











11

22




Acetylation

Phosphorylation

Methylation

Ubiquitination

Sumoylation

Citrullination

Ribosylation

Crotonylation

Biotinylation


23

Acetylation






P300

PCAF H2B


Ac Ac Ac Ac


H4

Ac H2A


Ac

H3


Ac Ac Ac Ac Ac

Ac Ac Ac Ac

12

24




treatment agents



33

Methylation

JHDM3 LSD1 PAD4

JHDM3

bull On Arg Lys

bull Reversible



Regulate

bull Cell cycle




13



Today In Press 2011

36

Phosphorylation

Main function

bullCell cycle

bullMeiosis

bullApoptosis


bullDevelopment

bullCell division

H2A


P

1

H2B


P

H3


P P P P


H4 P P

bull On Ser Thr

14

38












40





15

42














cell mitosis marker

Left anti-phospho

histone H2B (Ser14)

(07-191)

Right DAPI

44

Ubiquitinization


H2A(5-15)和H2B(1-15)






H2A

119

u

120

H2B


u

16

45

Sumoylation


Not hydrolysis

H2AH2BH4





46

Citrullination


bull MS


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


9

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

Chromatin Packaging


Histone octamer



ndash Nucleosome

H1 + Nucleosome

ndash 30 nm fiber






10

20

Histones

H1 H2A H2B H3 H4











11

22




Acetylation

Phosphorylation

Methylation

Ubiquitination

Sumoylation

Citrullination

Ribosylation

Crotonylation

Biotinylation


23

Acetylation






P300

PCAF H2B


Ac Ac Ac Ac


H4

Ac H2A


Ac

H3


Ac Ac Ac Ac Ac

Ac Ac Ac Ac

12

24




treatment agents



33

Methylation

JHDM3 LSD1 PAD4

JHDM3

bull On Arg Lys

bull Reversible



Regulate

bull Cell cycle




13



Today In Press 2011

36

Phosphorylation

Main function

bullCell cycle

bullMeiosis

bullApoptosis


bullDevelopment

bullCell division

H2A


P

1

H2B


P

H3


P P P P


H4 P P

bull On Ser Thr

14

38












40





15

42














cell mitosis marker

Left anti-phospho

histone H2B (Ser14)

(07-191)

Right DAPI

44

Ubiquitinization


H2A(5-15)和H2B(1-15)






H2A

119

u

120

H2B


u

16

45

Sumoylation


Not hydrolysis

H2AH2BH4





46

Citrullination


bull MS


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


10

20

Histones

H1 H2A H2B H3 H4











11

22




Acetylation

Phosphorylation

Methylation

Ubiquitination

Sumoylation

Citrullination

Ribosylation

Crotonylation

Biotinylation


23

Acetylation






P300

PCAF H2B


Ac Ac Ac Ac


H4

Ac H2A


Ac

H3


Ac Ac Ac Ac Ac

Ac Ac Ac Ac

12

24




treatment agents



33

Methylation

JHDM3 LSD1 PAD4

JHDM3

bull On Arg Lys

bull Reversible



Regulate

bull Cell cycle




13



Today In Press 2011

36

Phosphorylation

Main function

bullCell cycle

bullMeiosis

bullApoptosis


bullDevelopment

bullCell division

H2A


P

1

H2B


P

H3


P P P P


H4 P P

bull On Ser Thr

14

38












40





15

42














cell mitosis marker

Left anti-phospho

histone H2B (Ser14)

(07-191)

Right DAPI

44

Ubiquitinization


H2A(5-15)和H2B(1-15)






H2A

119

u

120

H2B


u

16

45

Sumoylation


Not hydrolysis

H2AH2BH4





46

Citrullination


bull MS


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


11

22




Acetylation

Phosphorylation

Methylation

Ubiquitination

Sumoylation

Citrullination

Ribosylation

Crotonylation

Biotinylation


23

Acetylation






P300

PCAF H2B


Ac Ac Ac Ac


H4

Ac H2A


Ac

H3


Ac Ac Ac Ac Ac

Ac Ac Ac Ac

12

24




treatment agents



33

Methylation

JHDM3 LSD1 PAD4

JHDM3

bull On Arg Lys

bull Reversible



Regulate

bull Cell cycle




13



Today In Press 2011

36

Phosphorylation

Main function

bullCell cycle

bullMeiosis

bullApoptosis


bullDevelopment

bullCell division

H2A


P

1

H2B


P

H3


P P P P


H4 P P

bull On Ser Thr

14

38












40





15

42














cell mitosis marker

Left anti-phospho

histone H2B (Ser14)

(07-191)

Right DAPI

44

Ubiquitinization


H2A(5-15)和H2B(1-15)






H2A

119

u

120

H2B


u

16

45

Sumoylation


Not hydrolysis

H2AH2BH4





46

Citrullination


bull MS


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


12

24




treatment agents



33

Methylation

JHDM3 LSD1 PAD4

JHDM3

bull On Arg Lys

bull Reversible



Regulate

bull Cell cycle




13



Today In Press 2011

36

Phosphorylation

Main function

bullCell cycle

bullMeiosis

bullApoptosis


bullDevelopment

bullCell division

H2A


P

1

H2B


P

H3


P P P P


H4 P P

bull On Ser Thr

14

38












40





15

42














cell mitosis marker

Left anti-phospho

histone H2B (Ser14)

(07-191)

Right DAPI

44

Ubiquitinization


H2A(5-15)和H2B(1-15)






H2A

119

u

120

H2B


u

16

45

Sumoylation


Not hydrolysis

H2AH2BH4





46

Citrullination


bull MS


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


13



Today In Press 2011

36

Phosphorylation

Main function

bullCell cycle

bullMeiosis

bullApoptosis


bullDevelopment

bullCell division

H2A


P

1

H2B


P

H3


P P P P


H4 P P

bull On Ser Thr

14

38












40





15

42














cell mitosis marker

Left anti-phospho

histone H2B (Ser14)

(07-191)

Right DAPI

44

Ubiquitinization


H2A(5-15)和H2B(1-15)






H2A

119

u

120

H2B


u

16

45

Sumoylation


Not hydrolysis

H2AH2BH4





46

Citrullination


bull MS


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


14

38












40





15

42














cell mitosis marker

Left anti-phospho

histone H2B (Ser14)

(07-191)

Right DAPI

44

Ubiquitinization


H2A(5-15)和H2B(1-15)






H2A

119

u

120

H2B


u

16

45

Sumoylation


Not hydrolysis

H2AH2BH4





46

Citrullination


bull MS


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


15

42














cell mitosis marker

Left anti-phospho

histone H2B (Ser14)

(07-191)

Right DAPI

44

Ubiquitinization


H2A(5-15)和H2B(1-15)






H2A

119

u

120

H2B


u

16

45

Sumoylation


Not hydrolysis

H2AH2BH4





46

Citrullination


bull MS


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


16

45

Sumoylation


Not hydrolysis

H2AH2BH4





46

Citrullination


bull MS


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


17



me2 me3

Ac

me3

Ac

Ph

Ac

me3


bivalent promoters


me3



- active genes


- mitosis




18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


18


Enhancers

BivalentSilence

Initiation

Elongation Desert

Heterochromati

n Insulator



TSS

UTR

Exons


LINEs SINEs

Heterochromatin

H3K4me1

AcH3K27

H3K9me3

H3K36me3 AcH3K9

AcH4

H3K4me3

Active Promoter

H3K27me3

Inactive Promoter

H3K36me3

H4K20me1

AcH3K9

Centromere

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


19

53

Pro

DNA







status

Chromatin IP (ChIP)


56

ChIP Workflow

N - + I









1

2

3

4

5

7

6

20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


20









bull Data Analysis



WB

IP

IFIHC

ChIP

Pre

dic

tive

Ch

IPa

bil

ity

59



Target

Abundance Target

Cells

ChIP

ChIPs

15 cm Plate






1

21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


21









mock IgG control)





002 004 003 003001 002

043

051

035038

028033

000

025

050

075

C-M

yc

B-G

lobin

Myo

D

GAPD

H In

tron

SYN R

E-1

GAPD

H C

oding

Pe

rce

nt

of

Inp

ut

001006

002

060

000

020

040

060

080

IgG TCF4 IgG TCF4

Pe

rce

nt

Inp

ut

GAPDH

Promoter

Sp5

Promoter


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


22

Fixation





of sonication






37degC)


weaker interactions




interactions


epitope


2








cell density



Sonication




Enzymatic Treatment

Nucleosomal

Ladder


Cat 17-375 EZ-Zyme





chromatin

TroubleShooting



3

23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


23

69

Antibody








ChIP validated Abs

4

70


Agarose Beads

More cost effective


(BSA carrier DNA)

Pre-clear samples


Magnetic Beads






throughput


can be problematic

5

24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


24






Improved Purity

Easier to handle




72






proteins


analysis

7

25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


25


Successful ChIP

Lower background

Specificity

Repeatable results




26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


26




No

ne

Un

mo

dif

ied

H3K

4m

e1

H3K

4m

e2

H3K

4m

e3

H3K

9m

e3

H3K

27m

e3

Single band on WB

Peptide Blocking




40 ng

4 ng

Dot Blot


Peptide Microarray






AbSurancetrade

Arrays

27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


27


Successful ChIP

Lower background

Specificity



79




28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


28

bull Speedy 14sec

bull Accurate


82









volume


bull DNA input

29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


29

83


17-295 ChIP Kit



17-371 EZ-ChIP

17-375 EZ-Zyme






1

2

3


Most citation



New












lysis reagents


tissues





30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


30


85


New


bull Less sample

- 104~105 cellswell

bull Less Antibody




- Protein AG blend

86

ChIP Ab+








31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


31



Enzymatic approach













Feature rich rack


Blocked for ChIP




Nucleosomal

Ladder

90














32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


32

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

92







coding RNAs


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


33

96



(pre-mRNA mRNA)

Protein Coding

Small Nuclear RNAs

(snRNA snoRNA)

Splicing

Ribosomal RNAs

(rRNA)


Transfer RNAs

(tRNA)




Non-coding RNAs







bull RNA splicing

bull Stability

bull Transport



34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


34

98
















35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


35

101

Transcription

Processing

Transport

Stability

Localization

Translation

Post-transcription

nucleus

cytoplasm

RNA-Binding Protein

Regulated (RIP)

DNA-Binding Protein

Regulated ( ChIP)

102



03-102 RIPAb+ HuR



03-105 RIPAb+ Lin28


03- 246 RIPAb+ FXR2






amp more 100

30469

0

50

100

150

200

250

300

350

400

IgG PABPC1

Fo

ld E

nri

ch

me

nt







36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


36

103




07-599 Anti-Ago 1

07-590 Anti-Ago 2










07-260 Anti-AUF1








07-717 Anti-Drosha

07-397 Anti-Ebp1












AB5969 Anti-HuB

AB5829 Anti-HuC

AB15882 Anti-HuC

AB5971 Anti-HuD

AB5831 Anti-HuR

07-1735 Anti-HuR

07-468 Anti-HuR




07-1385 Anti-Lin28


07-575 Anti-MDM2



AB5977 Anti-Musashi


07-637 Anti-Nova-1



AB15244 Anti-NXF2






AB3242 Anti-PGC-1








AB9904 Anti-QKI-5

AB9906 Anti-QKI-6

AB9908 Anti-QKI-7






AB5781 Anti-Staufen


104


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


37




Novel




bull Sensitive



Applications










38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


38

Agenda


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare










39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


39


bullImprinting


bullAging






How is it studied










resolution



40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


40



Sodium

Bisulfite

Conversion


cause its damage




MeDIP bull Fast




binding



Methylated

DNA-binding

Proteins




Methylation

Specific

Restriction

Enzymes

bull Easy to use


enzymes


(low resolution)




Applications by

Detection

Platform


Quantity by

Appplication

41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


41

113

MSP

BLAH U U U U U








mC hmC

42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


42










applications

0

2

4

6

8

Ho

urs



18

22

26

30

34

38

Cycle

Th

resh

old

M DNA 3365 3385





Non-CpG methylation



43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


43

119


MSP primer design




DNA Microarray



Methylated

DNA of interest

Downstream

Applications

DNA Microarray

DNA Sequencing


CpGenome

MethylQuest

Methyl-DNA

IP

Mag Bead

+

Mag Bead

Mag Bead

Mag Bead

44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


44

Summary


Chromatin



DNA Methylation




ndash ChIP

ndash Bisulfite

ndash RIP

ndash SmartFlare

128


45



Representative


130






















46

131

Questions

Merck Millipore


132


45



Representative


130






















46

131

Questions

Merck Millipore


132


46

131

Questions

Merck Millipore


132


gene regulation & epigenetics concepts and...

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