This document describes a digital reconstruction method called DigiWest that allows high-throughput analysis of Western blots. DigiWest separates proteins by molecular weight, cuts the membrane into strips, and elutes the proteins to couple them to color-coded Luminex beads. This allows multiplexed analysis using standard antibodies. The method was tested on liver tissue to analyze signaling pathways with higher sensitivity than traditional Western blots and allowed analysis of over 1,000 lanes of a kinase panel to study lapatinib resistance. DigiWest provides a quantitative digital output and increased throughput for protein signaling analysis.

1. Digital reconstructions of Western-Blots
Dhirendra K. Singh
Ph.D Student
Lab: Dr. Attila Gascer
Department of Microbiology
University of Szeged
Hungary
Journal Club Presentation

2. DigiWest: a high throughput Western-Blot and its application for comprehensive signaling
analysis of microdissected liver tissue
Dissertation
der Eberhard Karls Universität Tübingen
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
(Dr. rer. nat.)
vorgelegt von
Fridolin Treindl
aus Binsdorf
Tübingen
2015

4. Sample lysis (Cellls/tissues) Protein quantification assay
Loading and running
the gel
Transferring the protein from
the gel to the membrane
Antibody staining Picture
Analysis

5. Drawbacks
Western-Blot: limitations in the number of samples that can be
analyzed.

6. 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
LowsignalQuality
 Specificity of Antibodies
 Data processing and quality

7. 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

8. Biotinylation Cut horizontally into
strips
Place strips in
96-well plate
Elution buffer, bound
proteins are solubilizedAdd Neutravidin-coated
Luminex beads
Immobilize biotinylated
proteins on bead surfaces
Add Primary Ab and secondary antibody
for signal generation.

9. Biotinylation
Color code defines different molecular weight

10. 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

11. 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.

12. Protein elution from membrane
Organic solvents
Detergents, 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 glutamine
synthetase (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

13. 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.

14. 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 Analysis
Readout which is performed on a Luminex FlexMAP 3D

15. 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 plate
Analysis 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

17. First calculation
Subtraction of background signal
Options- Substract baclkgrounds- Subtracts a given quantile over
all measured wells for each bead-set
about +/- 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

18. 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

20. Characteristics of DigiWest.

21. Performance and output of DigiWest
fluorescenceintensity

22. Lapatinib resistance in a cell line model
Kinase inhibitor specifically inhibiting the receptor tyrosine kinases Her2 and
EGFR

23. 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

25. Volcano plot:p-value (ANOVA) < 0.001, fold
change > 2
24 significantly changed kinases was
analyzed using DigiWest and compared
to the MS analysis

26. 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.

27. Ingenuity Pathway Analysis- Points to central role
of p53

28. Regulatory Analysis:

29. 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)

30. Paired invasive (IDC) and ductal carcinoma in situ (DCIS)
Her2-stained sections, paired IDC and DCIS from three patients.
Her2 signals from DigiWest.

31.  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

32. Infection Microbiology
About my lab
Complement Protein
P.I.- Dr. Attila
Gacser
Associate Prof.
Post Doc:
Dr. Tibor Nemeth
Reneta Toth
Ph. D student:
Csaba Pap
Tanmoy Chakraborty
Erik Zjata
Dhirendra K. Singh
Sara
Mate Vadovics
Trainee Students:
6+ M.Sc and B.Sc
Candida
parapsilosis
Molecular
Biology
Immunology
Innate immunity
Adaptive immunity
Aspartic
Proteases
Knock Down and
Overexpresion of genes

33. Conclusion
Thumbs Up

34. Questions??????
Remarks……...