Serving Your Proteomics Needs
* 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)
|Red: Drug treated
Green: Normal / Red: Drug treated
Blue: Phospho-protein staining
Green: Normal / Blue: Phospho-staining
Normal / Treated / Phosphorylated
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
After de-staining the gel, scan the gel image of the phosphorylated proteins
5. In-gel Analysis of Differential Protein
Proteins that change in abundance between samples were identified in-gel by DeCyder software
6. Locate the Phospho-protein Spots on 2D
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
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.
Goal: To identify global phosphorylation changes between 2 or more samples
Drug Treatment: Phospho-Spots
Control Protein / Phospho-Spots
in Control Sample
Drug Protein / Phospho-Spots
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.
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.