A. 2D DIGE Followed by Phosphorylated-protein
Expression Profiling
B. Total Protein versus Phosphorylated-Protein
Expression on 2D Image
C:
2D Phosphorylated-Western Blot
A.
2D DIGE Followed by Phosphorylated-protein Expression Profiling
Goal: Identify differentially expressed
proteins that are phosphorylated
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Example: 2D DIGE followed by
phosphorylated-protein expression profiling
Protein extracts from mouse liver (before and after
drug treatment) were labeled by minimal CyDyes respectively,
followed 2D DIGE (pH 4-7) procedure. Phosphorylated-protein
image was scanned after fluorescent phosphorylated-protein
staining. The data illustrates:
1. Proteins differentially expressed between ‘normal’
and ‘treated’ (image 1+2 overlay)
2. Phosphorylated proteins in either ‘normal’
and/or ‘treated’ sample (image 3)
3. Phosphorylated proteins that are increased, decreased
or similar in abundance between ‘normal’
and ‘treated’ (image 1+2+3 overlay)
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Green:
Normal
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Red:
Drug treated
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Green:
Normal / Red: Drug
treated
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Blue: Phosphorylated-protein staining
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Green:
Normal / Blue: Phosphorylated-staining
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Normal
/ Treated /
Phosphorylated
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1. 2D DIGE Protein Expression Profiling
2D DIGE gel is run as described before (2D
DIGE procedure), followed by image scan using Typhoon
image scanner.
2. Gel Fixation
The 2D DIGE gel is fixed overnight with fixation solution.
3. Phosphorylated-protein Fluorescent Staining
Stain the gel with fluorescent phospho-protein staining
solution
4. Phosphorylated-imaging
After de-staining the gel, scan the gel image of the phosphorylated
proteins
5. In-gel Analysis of Differential Protein Expression
Proteins that change in abundance between samples were identified
in-gel by DeCyder software
6. Locate the Phosphorylated-protein Spots on 2D Images
Phospho-protein spots can be located by in-gel overlaying
the 2D DIGE and the Phospho-protein Image using ImageQuant
software
7. Quantitation of Differentially Expressed Phosphorylated-protein
For those phosphorylated protein spots, in-gel analysis
of their protein fold change can be obtained using DeCyder
software
8. Data Report
In addition to a typical 2D DIGE report, phosphorylated
protein spots are circled and numbered, and their quantitative
change in abundance is documented by DeCyder software.
B.
Total Protein versus Phosphorylated Protein on 2D Image
Goal: To identify global phosphorylation
changes between 2 or more samples
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Control:
Phosphorylated-Spots
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Drug Treatment:
Phosphorylated-Spots
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Control Protein / Phosphorylatead-Spots
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Drug Protein / Phosphorylated-Spots
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Increased Phosphorylation
in Control Sample
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Increased Phosphorylation
in Drug Treated Sample
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1. 2D Gel
Each sample is labeled with a CyDye and run on 2D gels to
separate the proteins by pI and MW, followed by image scan
using Typhoon image scanner.
2. Gel Fixation
The gels are fixed overnight using fixation solution.
3. Phosphorylated-protein fluorescent staining
Stain the gels with fluorescent phosphorylated-protein staining
solution.
4. Phosphorylated-imaging
After de-staining the gels, scan the gel images of the phosphorylated-proteins.
5. Image Overlay and In-gel Analysis
Phosphorylated-protein spots can be located by overlaying
the Phosphorylated-protein Image and total protein image
using ImageQuant software.
6. Data Report
Differences in phosphorylation pattern are identified between
two or more comparisons. The report will includes gel images
with differentially phosphorylated spots circled and numbered.
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Phosphorylated
Protein Profiling
