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A. 2D DIGE Followed by Phospho-protein Expression Profiling

B. Total Protein versus Phospho-Protein Expression on 2D Image

C: 2D Phospho-Western Blot

* Example: 2D DIGE followed by phospho-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
Red: Drug treated
Phospho-Protein Profiling -green :normal
Phospho-Protein Profiling  - red : drug treated
Green: Normal / Red: Drug treated
Phospho-Protein Profiling - overlay

Blue: Phospho-protein staining
Phospho-Protein Profiling  - blue staining
Green: Normal / Blue: Phospho-staining
Normal / Treated / Phosphorylated
Phospho-Protein Profiling  - green: normal / Blue: phospho staining
Phospho-Protein Profiling  - Phosphorylated


A. 2D DIGE Followed by Phospho-protein Expression Profiling (see an example)

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 fold change in abundance 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

1. 2D Gel
2D gel was run to separate the protein by PI and Mw.

2. Gel Fixation
The gel is fixed overnight with fixation solution.

3. Phospho-protein fluorescent staining
Stain the gel with fluorescent phosphor-protein staining solution.

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

5. Removing Phospho-protein Fluorescent Staining
Removing the phospho-protein staining to prepare the gel for total protein staining.

6. Total Protein Staining
Stain the gel again with Deep Purple fluorescent stain (GE Healthcare) or Sypro Ruby (Invitrogen).

7. Total Protein Imaging
After destaining the gel, scan the gel image of total proteins.

8. 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. The in-gel analysis of the phospho-protein is performed using DeCyder software.

9. Data Report
Includes gel images with phosphorylated protein spots circled and numbered.

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