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……...
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