DigiWestDigital reconstructions of Western-

Blots

Dhirendra K. SinghPh.D StudentLab: Dr. Attila GascerDepartment of MicrobiologyUniversity of SzegedHungary

Journal Club Presentation

DigiWest: a high throughput Western-Blot and its application for comprehensive signaling analysis of microdissected liver tissue

Dissertationder Eberhard Karls Universität Tübingen

zur Erlangung des Grades einesDoktors der Naturwissenschaften

(Dr. rer. nat.)vorgelegt von

Fridolin Treindlaus Binsdorf

Tübingen2015

Sample lysis (Cellls/tissues) Protein quantification assay Loading and running the gel

Transferring the protein from the gel to the membrane

Antibody staining Picture

Anal

ysis

DrawbacksWestern-Blot: limitations in the number of samples

that can be analyzed.

Protein microarray Reverse phase protein microarrays (RPPMs)

Western-Blot GAP

Less samples

Western-Blot method

+Allow

generation of many copies or replicas of the

blot

DigiWest

Low

sig

nal Q

ualit

y

Specificity of Antibodies Data processing and quality

a) Separation of proteins by molecular weight- classical SDS-PAGE and Blotting the proteins onto a membrane.

c) Cutting the membrane in pieces, containing the separated proteins of a certain molecular weight range and elution of the proteins.

d) Coupling of the proteins from each molecular weight fraction onto distinct, color coded sets.

e) Incubation of aliquots of the pooled bead-mixes using standard Western-Blot antibodies followed by fluorescent secondary antibodies for Luminex readout.

g) Analysis of the Luminex signals.

Digi West: Principle

Biotinylation

Cut horizontally into strips

Place strips in 96-well

plate

Elution buffer, bound proteins are solubilized

Add Neutravidin-coated Luminex

beads

Immobilize biotinylated proteins on bead surfaces

Add Primary Ab and secondary antibody for signal generation.

Biot

inyl

atio

n

Color code defines different molecular weight

Method Development Preparation of samples

Western-Blotting Protein biotinylation

Efficient loading of the proteins onto the streptavidin coated beads will be done by biotinylation of protein.

Biotinylation BSA or milk powder

Tween-20

NHS-LC-LC-Biotin

Washing

Cutting membranes

Ruler and scalpel: cut into molecular weight fractions.

Smaller fractions Improved the resolution of the data Increased sensitivity and to a reduced background. Increase of the overall binding capacity.

Protein elution from membraneOrganic solventsDetergents, usually in alkaline buffers or even in a moderate sodium hydroxide solution or Acetone adding an aqueous buffer.

400 µg mouse liver lysate Detergent-Mix• 0.4% PVP 10000 • 0.4% PVP 40000 • 0.7% Triton X100 & 0.5%

Tween20• Detergent-Mix Urea• Detergent-Mix with 4 M Urea• 1% Triton 8 M Urea• 1% Triton X100 and 8 M Urea• 1% Triton 4 M Urea• 1% Triton X100 and 4 M Urea• 1% Triton’ 1% Triton

Areas surrounding the position of glutaminesynthetase (42 kDa) were cut from each membrane, 4.5 mm high and 10 mm wide, biotinylated, and again cut into 5 snippets each 2 mm wide for elution under 5 different conditions

Loading proteins onto Luminex beads

NeutrAvidin (pI 6.3)

Streptavidin (pI 4.6)similar affinity for biotin (Kd 10-15 M)

NeutrAvidin showed a better overall performance

Luminex beads: Polystyrene or paramagnetic microsphere beads, internally dyed with different flurophore of different intensities.

Summary of the workflow Separation of protein and blotting. Washing of membrane with PBST and a Ponceau S

to visualize the transferred proteins. Label lanes using a pencil before the blots. Biotinylation of membrane by PBST + NHS-PEG12-

Biotin. Membrane cutting put in 96 well plates and elute

by adding 10 µl of the elution buffer. Eluted proteins are diluted by adding 90 µl BSA. Transferred to bead plates contain a defined

number of beads per well. Primary antibodies Labelled secondary antibodies.

DATA AnalysisReadout which is performed on a Luminex FlexMAP

3D

Data analysis Luminex output file contains several blocks of

data. Luminex uses a relative fluorescence unit called

MFI (median fluorescence intensities). 96 wells per plate and up to 384 bead sets

resulting in almost 37000 data points per plateAnalysis tool

3 visible sheets:

First sheet is meant for input of the Luminex data

Second is for data analysis. Third contains the quantified signals from the

selected peak

First calculationSubtraction of background signalOptions- Substract baclkgrounds- Subtracts a given quantile over all measured wells for each bead-setabout +/- 5 MFI for stable measurements, about +/- 10 MFI for less stable ones).

Peaks are selection:

When the molecular weight of the analyte is entered, the tool searches for the peak (local maximum) in a definable area around this molecular weight.

Calculation

QUALITATIVE COMPARISON OF WESTERN-BLOT AND DIGIWEST

20 µg HepG2 lysate were used to generate a DigiWest

40000 beads

96 molecular weight fractions 200 antibody

DigiWest data which were compared to the corresponding Western-Blot images

Characteristics of DigiWest.

Performance and output of DigiWestflu

ores

cenc

e in

tens

ity

Lapatinib resistance in a cell line modelKinase inhibitor specifically inhibiting the receptor tyrosine

kinases Her2 and EGFR

Digi West : Detection of cellular signals Mucoepidermoid pulmonary

carcinoma cell line H292. Question: Identify the change in cell signaling differences that occur during development of resistance to Lapatinib using kinases?

ATP

Protein profiling

Volcano plot:p-value (ANOVA) < 0.001, fold change > 2

24 significantly changed kinases was analyzed using DigiWest and compared to the MS analysis

Volcano plot of the Kinobead pull-down analysis. Lapatinib-resistant H292 cell line compared with the

parental cell line. The data contained 1,000 western blot lanes. A total of 185 analytes were detected, including 74 phosphorylations.

Ingenuity Pathway Analysis- Points to central role of p53

Regulatory Analysis:

Protein expression analysis in primary tumour cells: Protein analysis of microdissected material is

challenging due to limited amount of protein. DigiWest technology ???????

Primary tumour cells mammary carcinoma tissue

Laser-capture microdissection

DigiWest protein profiling

Invasive ductal carcinoma (IDC)and Ductal carcinoma in situ (DCIS)

Paired invasive (IDC) and ductal carcinoma in situ (DCIS)

Her2-stained sections, paired IDC and DCIS from three patients.

Her2 signals from DigiWest.

Reduced sample requirement and detection quantitative phospo status.

No external molecular weight markers, only intrinsic markers.

Increased throughput-Run 60-600 analysis per sample with 2-60 samples per study.

Automated workflow and digital output.

Play with data: easily accessible graphical format.

Converting obtained signal intensities to a greyscale image.

Smoothing the image using a Gaussian filter results in a western blot-like data representation.

Conclusion:Digi West

Infection Microbiology

About my lab

Complement Protein

P.I.- Dr. Attila Gacser

Associate Prof.

Post Doc: Dr. Tibor NemethReneta Toth

Ph. D student:Csaba PapTanmoy ChakrabortyErik ZjataDhirendra K. SinghSara Mate Vadovics

Trainee Students:6+ M.Sc and B.Sc

Candida parapsilosis

Molecular Biology

Immunology

Innate immunityAdaptive immunity

Aspartic Protease

s

Knock Down and Overexpresion of genes

Conclusion

Thumbs Up

Questions??????Remarks……...