sample prep – the easy waycgs.hku.hk/portal/files/grc/events/seminars/2010/20101021/sample... ·...

Sample prep – the easy way

Johan Öhman, Senior ScientistGE Healthcare, Uppsala, Sweden

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Content

Introduction

Homogenization and Stabilization

Extraction and Solubilization

Enrichment, depletion and fractionation

Enrichment of tagged proteins

Buffer exchange, concentration and clean-up

Summary

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Sample preparation - from sample to analysis

Analytical resultsSample source

Buffer exchange, concentration, clean up

Ensure compatibility in work flow

Collect sample and extract/stabilize

proteins


Protein analysis

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Why focus on the preparation of the samples ?

Quality of the results of the analysis• Reproducibility – correct comparison between the biological events in different samples

• Yield – as high recovery as possible will enable more comprehensive analyses to be made

• Purity – removing the contaminants will greatly enhance the conclusiveness of the analyses

Time savings• Spend time on analyzing only relevant data

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Breaking up cells and tissueSample Grinding Kit

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Sample Grinding Kit•Disrupts small tissue and cell samples for protein extraction.•Uses 1.5 ml microcentrifuge tubes, grinding resin, and disposable pestles.•Process up to 100 mg of sample per tube in about 10 min.

Samples treated : 50Sample weight : 100 mgTime : 10 minStorage : R.T.Shelf life : unlimited

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Protein StabilizationProtease Inhibitor Mix

PlusOne™ Reagents

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Protease Inhibitor Mix

• Protease inhibitor cocktail specifically developed for sample preparation in 2-D studies.

• No EDTA is used, which allows optimal nuclease activity for removing nucleic acids from samples.

• Contains a mixture of both competitive and noncompetitive protease inhibitors that inhibit serine, cysteine, and calpainproteases. Optionally, EDTA may be added to inhibit metalloproteases.

• Optimized concentration for excellent inhibition of protease activities. Effectively inhibits over 95% of protease activity.

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PlusOne™ Reagents

• EDTAInhibit metalloproteinases by chelating free metal ions required for activity.Scavenges oxidation mediated by free radicals catalyzed by transition metal ions.

• DTTReducing agents are frequently included in the sample solution to break any disulfide bonds present and to maintain all proteins in their fully reduced state. The most commonly used reductant is dithiothreitol (DTT).

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Extraction and Solubilization

Extraction Native conditionsMammalian Protein Extraction Buffer

Extraction Denaturing conditions2-D Protein Extraction Buffer

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Mammalian Protein Extraction Buffer

A convenient method to extract total soluble protein from mammalian cultured cells.

A mild detergent-based buffer, eliminating the need for mechanical cell lysis, and delivering high quality protein lysate compatible with most downstream applications.

Components in the buffer: 500 ml

<20mM Tris-HCl, <20mM NaCl, <5% NP-40, <5% Triton X-100, <5% Tween 20


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Key features and benefitsMammalian Protein Extraction Buffer

• Ready to use buffer • Mild and stable formulation – high recovery• Maintained biological activity • Reproducible results• Compatible with most downstream applications

• Complete declaration of buffer components

• Saves Time


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Mammalian Protein Extraction Buffer: Reproducible cell lysis with maintained high protein activityA: High reproducibility with Mammalian Protein Extraction Buffer9 individual samples in orange from 107 CHO-cells in 0.3 ml.

B: High protein carbonic anhydraseactivity in CHO cell extract prepared with Mammalian Protein Extraction Buffer (orange) or in a homemade RIPA* buffer (blue). The average relative activity and standard deviation is shown . The figure shows that high protein activity was retained using both methods.

*50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, and 0.1% Na-deoxycholate

A

B


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2-D Protein Extraction Buffer Trial Kit and 2-D Protein Extraction Buffer-I,-II, -III, -IV, -V and VI

Buffers are designed to produce high spot resolution for 2-D gel analysis, and is a convenient method to prepare high quality total protein from cells and tissues

Easy optimization of total protein extraction with 2-D Protein Extraction Buffer Trial Kit.

Each of the six buffers in the trial kit are available in larger pack size once the optimal buffer for your specific sample has been determined.

Buffers can also be used prior to 1-D electrophoresis or for rehydrating IPG strips prior to 2-D electrophoresis


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Protein map differences between 2-D Protein Extraction Buffers

2-D DIGE gel image of a mouse liver sample extracted using Extraction Buffer-II overlaid on a sample extracted by Extraction Buffer–VI


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Conclusions

The first steps in any protein analysis workflow determines the quality of the end results.

GE Healthcare has several sample preparation products for solubilization and stabilization of proteins from various source materials.

Reproducible results are obtained by using pre-made extraction buffers.


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proteins


Protein analysis


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Introduction – increasing detectability of targeted proteins

Protein fractionationDividing of an initial solubilized protein population into multiple sub-samples/fractions based on differences in non-specific properties of the present proteins.

Affinity based enrichment of protein

Affinity binders (ligands) with specific affinity for a particular group of proteins are immobilized to a solid support and used to bind the targeted group while letting all other proteins end up in flow-through and/or wash fractions.

Affinity based protein depletionAffinity binders are used to specifically bind unwanted proteins from a complex protein solution. Normally, only the flow-through fraction is passed on to analysis.


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Affinity based enrichment of proteins

Immunoprecipitation• SpinTrapTM

• MultiTrapTM

• Mag SepharoseTM

Work flow - example


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Comparison of recovery and purity between Protein A Mag Sepharose™and Dynabeads™ Protein A– a benchmark study


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Experimental design benchmark studyComparison of recovery and purity between Protein A Mag Sepharose™, GE Healthcare and Dynabeads™ Protein A, Invitrogen

Medium: 25 µl Protein A Mag Sepharose (GEHC) 50 µl Dynabeads™ Protein A (Invitrogen)

Sample: Capture of human transferrin froma complex sample

Protocol: The protocol for respective product was followed

Work flow


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Results benchmark studyPu

rity

(%)

Reco

very

(%)

Protein A Mag SepharoseTM, GEHC

DynabeadsTM Protein A, Invitrogen

Higher recovery with Protein A Mag Sepharose than with Dynabeads Protein A

- Paramagnetic beads with cross-linked agarose

- Visible, dense and non-adherent

MagRack 6


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Pull-Down

SpinTrapTM

Format

MagSepharoseTM

Microcentrifuge MagRack 6

• µg-mg scale• Fixed amount of beads• Fixed maximum sample volume

• Low µg scale-µg scale• Variable amount of beads• Variable sample volume• Automatable

NHS Mag Sepharose™NHS HP SpinTrap™

Products

Affinity chromatography medium designed for covalent coupling of any protein or biomolecule containing free amine groups

• Protein

• Peptide

• Substrate

• Inhibitor

• Antibody

Etc.

Sepharose bead +

NHS activated agarose resin

Sepharose bead

Covalently coupled protein/biomolecule


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Techniques for enrichment of phosphoproteins and phosphopeptides

Sample mixture

Immunoprecipitation Affinity matrix

pTyr-antibody

pSer-/pThr-antibody

IMAC MOAC

TiO2Fe3+ Ga3+

Al3+Zr4+


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Protein depletion

DepletionAlbumin & IgG Depletion SpinTrap™

HiTrap™ Albumin & IgG Depletion


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Depletion of human albumin and IgG from human plasma

• Detection of more proteins with similar isoelectric point and Mw as albumin and IgG

• Detection of more low abundance proteins

Undepleted plasma Depleted plasmaUndepleted plasma Depleted plasma


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Conclusions

Enrichment is necessary to detect low abundant non-tagged proteins.

GE Healthcare has a broad range of general sample preparation products for signal enhancement of target molecule.

The Mag Sepharose™ platform and the Trap platform is particularly suited for capture of small amounts of target molecules.


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proteins


Protein analysis


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Tagged protein purification

Enrichment

His SpinTrap™ / His SpinTrap Kit

His GraviTrap™


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Formats used in screening of Histidine tagged proteins

SpinTrapTM MultiTrapTM Gravity columns

Format

Microcentrifuge Centrifuge/vacuum Gravity

His SpinTrap

His SpinTrap Kit

His MultiTrap FF

His MultiTrap HP

His Buffer Kit

His GraviTrap™

His GraviTrap Kit


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His SpinTrap™ / His SpinTrap Kit• Ni Sepharose™ High Performance in prepacked spin columns.

• For simple, small-scale purification of histidine-tagged proteins and rapid expression screening.• Yields up to 750 μg pure protein per column.• Use manually with a centrifuge.


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His SpinTrap™ – four-stage, 10 min purification

1. Equilibration 2. Sample loading 3. Wash 4. Elution


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Application example using His SpinTrap™

Direct application of unclarified lysate- load of unclarified and clarified cell lysates for comparison of purity


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His SpinTrap™ - purification directly from unclarified cell lysates

1. LMW markers

2. Unclarified sample, start material (diluted 1:10)

3. Clarified sample, start material (diluted 1:10)

4. Unclarified sample, eluted pool

5. Clarified sample, eluted pool

Target protein:

GFP-(His)6


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His GraviTrap FF

Lane 1. Start material (diluted 1:10)Lane 2. Flowthrough (diluted 1:10)Lane 3. WashLane 4. Eluate

1 2 3 4

1 2 3 4

Unclarified sample — two steps

Clarified sample — many steps

His GraviTrap™


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Conclusions

GE Healthcare has many different gel media to purify tagged proteins reproducibly and with high yield.

GE Healthcare also supplies the gel media in many different formats and scales for convenience.


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proteins


Protein analysis


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Introduction - Ensure compatibility in work flow

• Buffer exchange and desalting- Manipulation of the buffer system

• Concentration and volume reduction- Adjust volume and global protein concentration

• Sample Clean-Up- Removal of non-protein contaminants and protein aggregates


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Buffer Exchange and Desalting

Gel filtrationPD family products


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PD family productsEfficient desalting and buffer exchange

Efficient removal of unincorporated labels

SpinTrapTM MultiTrapTM Gravity columns

Format

Microcentrifuge Centrifuge Gravity/centrifuge

PD SpinTrap™ G-25 PD MultiTrap™ G-25 PD MidiTrap™ G-25PD MiniTrap™ G-25PD MidiTrap G-10PD MiniTrap G-10PD 10 Desalting Column

Gel filtration medium:

- Sephadex™ G-25 medium, exclusion limit > 5000

- Sephadex G-10, exclusion limit > 700


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PD family

• Efficient small-scale removal of protein, peptides and carbohydrates

• Rapid parallel buffer exchange, desalting, and removal of small contaminants

• Broad product range for sample volumes: 70 µl up to 2.5 ml


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Removal of NaCl from bovine serum albumin

> 99> 95C) PD-10 Desalting Columns

> 9895B) PD MidiTrap™G-25

> 9995A) PD MiniTrap™G-25

Desalting capacity (%)

Recovery (%)

Column

High desalting capacity and recovery of target protein with gravity flow protocol

Sample: 1000 µg/ml bovine serum albumin (BSA) in 1 M NaCl

Sample volumes: A) 500 µl, B) 1000 µl and C) 2.5 ml


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Buffer exchange and desaltingconcentration and volume reduction

Diafiltration and ultrafiltrationVivaspin™ family products


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Vivaspin™ products

• Concentrates biological samples byultracentrifugation through aMembrane

• Separates biomolecules on the basisof size

• No risk of concentrating to dryness

• A gentle, non-denaturing method

• High recoveries, >95 %


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Sample volumes from 100 µl-20 mlAll products are available with MWCO values of:3000, 5000, 10 000, 30 000, 50 000 and 100 000

2 – 6 ml 5 – 20 ml400 µl – 2 ml100 – 500 µl

Vivaspin™ 500 Vivaspin 20Vivaspin 6Vivaspin 2

Volume range:


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PD vs. Vivaspin products

+ ++ +

++ +

+ +0DesaltingConc.

PD

Vivaspin™

PD and Vivaspin


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Sample Clean-Up

Precipitation

PlusOne™ SDS-PAGE Clean-Up Kit

Ettan™ 2-D Clean-Up Kit


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Ettan™ 2-D Clean-Up Kit• Improves the quality of 2-D electrophoresis results by removing interfering contaminants. • Quantitative precipitation separates proteins from detergents, salts, lipids phenolics, and nucleic acids.

PlusOne™ SDS-PAGE Clean-Up Kit• Prepares samples for SDS-PAGE that are otherwise difficult to analyze because of salts or low protein concentrations. • Quantitatively precipitate proteins while leaving interfering substances in solution.

2-D Clean-Up Kit eliminates horizontal streaking caused by residual SDS.


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Sample Clean-Up

ClarificationWhatman SPARTAN™ syringe filters


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Whatman SPARTAN™ syringe filters

Features• Ready-to-use filter unit with a hydrophilic, low protein-binding membrane made of regenerated cellulose

Applications• Filtration of organic and aqueous solutions in HPLC with reproducible results

• Purification of aqueous and organic solutions

• Filtration of protein solutions


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Summary

• GE Healthcare has products supporting each unit operation; buffer exchange and desalting, concentration/volume reduction and sample clean-up.

• General sample preparation tools that are needed in almost every laboratory.

• High quality products with a good technical support.

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Questions?

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proteins


Protein analysis

Each step in a workflow is important !

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MiniTrap, MidiTrap, MultiTrap, GraviTrap, SpinTrap, Sepharose, HisTrap, HiTrap, Ettan, PlusOne and SPARTAN are trademarks of GE Healthcare companies. GE, imagination at work and GE monogram are trademarks of General Electric Company.

All third party trademarks are the property of their respective owners.

Purification and preparation of fusion proteins and affinity peptides comprising at least two adjacent histidine residues may require a license under US pat 5,284,933 and US pat 5,310,663 , including corresponding foreign patents (assigne: Hoffman La Roche, Inc).

All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information.

© 2010 General Electric Company – All rights reserved.First published October 2010.

GE Healthcare Bio-Sciences AB, a General Electric Company.

GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden.

sample prep – the easy waycgs.hku.hk/portal/files/grc/events/seminars/2010/20101021/sample... ·...

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