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Phosphorylated-Protein Expression Profiling

 

 

 

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

* 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. Phospho-protein image was scanned after Fluorescent phospho-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)

Green: Normal

Phosphorylated-Protein Expression Profiling
Red: Drug treated

Phosphorylated Protein Expression Profiling

Green: Normal / Red: Drug treated

Phosphoprotein

Blue: Phospho-protein staining

Phospho-protein


Green: Normal / Blue: Phospho-staining

Phosphorylated-Protein Expression Profiling
Normal / Treated / Phosphorylated

Phosphoprotein Array


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. Phospho-protein Fluorescent Staining
Stain the gel with fluorescent phospho-protein staining solution

4. Phospho-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 Phospho-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 Phosphor-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

Control: Phospho-Spots

Total Protein versus Phosphorylated Protein on 2D Image 


Drug Treatment: Phospho-Spots

Total Protein versus Phosphorylated Protein on 2D Image

Control Protein / Phospho-Spots

Total Protein versus Phosphorylated Protein on 2D Image

Increased Phosphorylation
in Control Sample
Drug Protein / Phospho-Spots

Total Protein versus Phosphorylated Protein on 2D Image

Increased Phosphorylation
in Drug Treated Sample

 

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. Phospho-protein fluorescent staining
Stain the gels with fluorescent phosphor-protein staining solution.

4. Phospho-imaging
After de-staining the gels, scan the gel images of the phospho-proteins.

5. Image Overlay and In-gel Analysis
Phospho-protein spots can be located by overlaying the Phospho-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.