63
Appendix
Additional Staining Protocols
Pro-Q
®
Diamond
Phosphoprotein
Gel Stain
Instructions for specific staining of phosphoproteins on E-PAGE
™
Gels using
Pro-Q
®
Diamond Phosphoprotein Gel Stain is described in this section.
The total staining time is ~5 hours.
Due to the thickness of E-PAGE
™
Gels, the Pro-Q
®
Diamond Phosphoprotein
Gel Stain
may not be optimally sensitive
for your particular staining
application.
Materials Needed
You will need the following items for staining one E-PAGE
Gel:
•
Pro-Q
®
Diamond Phosphoprotein Gel Stain
•
Fixing solution (50% methanol, 10% acetic acid, made with deionized
water)
•
Pro-Q
®
Diamond Phosphoprotein Gel Destaining solution (or use 50 mM
sodium acetate pH 4.0 containing 20% acetonitrile as destaining solution)
•
Deionized water
•
Microwave safe
staining tray
•
Rotary shaker
•
Microwave oven (700–1000 watts)
•
UV transilluminator equipped with a standard camera or laser scanners
See page 71 for ordering information.
Phosphoprotein
Staining Protocol
For all steps described below, be sure to use sufficient volume of reagent to
completely cover the gel using a suitable container such that the gel moves
freely.
1.
After electrophoresis, remove gel from the cassette (page 24) and place the
gel in a clean
microwave saf
e staining container.
2.
Fix the gel in fixing solution (50% methanol, 10% acetic acid in deionized
water) for 45 minutes at room temperature with gentle shaking. Decant the
fixing solution and add fresh fixing solution to the container and repeat the
fixing step again for 45 minutes at room temperature. Decant the fixing
solution.
3.
Add deionized water to the gel and microwave at high power for
45 seconds. Remove the gel from the microwave and wash the gel for
10 minutes at room temperature with gentle shaking. Decant the water and
repeat the wash step again with deionized water. Decant the water.
4.
Stain the gel with Pro-Q
®
Diamond Phosphoprotein Gel Stain. Incubate the
gel in the
DARK
for 1–2 hours at room temperature with gentle shaking.
Decant the stain.
Summary of Contents for E-PAGE Gels
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