42
Coomassie Staining,
Continued
The staining solutions for the Coomassie R-250 staining protocol and the
microwave staining protocol are different. Be sure to use the correct stain for the
correct protocol.
Coomassie
®
R-250 Staining
Protocol
For all staining and destaining steps described below, be sure to use sufficient
reagents to completely cover the gel using a suitable container such that the gel
moves freely during the staining and destaining steps.
1.
After electrophoresis, remove the gel from the cassette (page 24) and place
the gel in a clean incubation tray.
2.
Prepare Coomassie stain (0.03% Coomassie R-250 in 30% methanol and 10%
acetic acid). See note on previous page.
3.
Stain the gel in the prepared stain for 1.5 hours at room temperature with
gentle shaking.
4.
Destain the gel in destaining solution (see previous page) at room
temperature with gentle shaking with intermittent changes of solution until
the bands are visible or overnight for maximum sensitivity and clear
background.
Coomassie R-250
Staining
Microwave
Protocol
For all staining and destaining steps described below, be sure to use sufficient
reagents to completely cover the gel in a
microwave safe
container such that the
gel moves freely during the staining and destaining steps.
1.
After electrophoresis, remove the gel from the cassette (page 24) and place
the gel in a clean
microwave safe
container.
2.
Prepare Coomassie stain (0.015% Coomassie R-250 in 30% ethanol and 10%
acetic acid). See note on previous page.
3.
Add enough stain to completely cover the gel in the
microwave safe
container.
4.
Warm the gel and solution to about 50°C in a microwave oven (see page
41).
Note:
Do NOT boil the solution.
5.
Incubate the gel in the warmed staining solution for 30 minutes on an
orbital shaker at room temperature.
6.
Discard the stain, rinse the gel briefly with water and discard the water.
7.
Add enough destaining solution (see previous page) to cover the gel during
incubation
8.
Place two pieces of positively charged nylon membrane on top of the
destaining solution to speed up the destaining process.
9.
Warm the destaining solution, gel and nylon membrane to 50°C in a
microwave oven (see previous page).
Note:
Do NOT boil the solution.
10.
Incubate the gel in the warm destaining solution on an orbital shaker at
room temperature until the desired background is achieved.
Note:
To obtain a clear background, perform destaining overnight. Results
obtained with Coomassie stain are shown on page 56.
Summary of Contents for E-PAGE Gels
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