A. 2-D DIGE Followed by Phospho-protein
Profiling
B. Total Protein versus Phospho-Protein
on 2-D Image
*
Example: 2-D DIGE followed by
phospho-protein profiling
Protein extracts from mouse liver (before and after
drug treatment) were labeled by minimal CyDyes respectively,
followed 2-D 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
|
|
|
|
|
|
|
Green:
Normal / Red: Drug
treated
|
|
|
|
Blue: Phospho-protein staining
|
|
|
|
|
|
Green:
Normal / Blue: Phospho-staining
|
Normal
/ Treated /
Phosphorylated
|
|
|
|
A.
2-D DIGE Followed by Phospho-protein Profiling (see
an example)
1. 2-D DIGE Protein Profiling
2-D DIGE gel is run as described before (2-D
DIGE procedure), followed by image scan using Typhoon
image scanner.
2. Gel Fixation
The 2-D 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 2-D Images
Phospho-protein spots can be located by in-gel overlaying
the 2-D 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 2-D 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 2-D Image
1. 2-D Gel
2-D 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.
