Bio-Rad ReadyPrep™ 2-D Starter Kit User Manual
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4.9 STAINING.
The 2-D starter kit was designed so that sufficient protein sample loaded in the IEF
dimension readily stain second-dimension SDS-PAGE gel with widely available Coomassie
Blue stains such as Bio-Rad’s Bio-Safe Coomassie stain and Coomassie Brilliant Blue R-
250 stain. These staining protocols are rapid and easy to perform so that the 2-D elec-
trophoresis results can be quickly assessed. A description of each staining protocol is pro-
vided below.
1. BioSafe Coomassie Stain.
a
Fill enough staining trays with nanopure water for the number of gels run and set
aside (for Ready Gel and Criterion gels use 200 ml per gel and for PROTEAN II Ready
Gel gels use 400 ml per gel).
b
At the conclusion of the SDS-PAGE, open each gel cassette and place each gel into a
tray with water.
c
Wash the gels 3 times for 5 minutes each. Add fresh water for each wash.
d
Add enough Bio-Safe stain to completely cover each gel. For Ready Gel and Criterion
gels about 50 ml per gel is sufficient. For PROTEAN II Ready Gel gels use about 100
ml of the stain.
e
Place each gel on a rocker or orbital shaker and shake for at least 60 minutes. The
gels can be left in the stain overnight if desired.
f
Discard the stain and wash the gels twice for 15-30 minutes with water. Longer water
washes may be needed to remove remaining background. The gels can be stored in
water for several days.
g
Figure 14 shows the expected 2-D gel images of the E. coli protein sample run on
each of the 3 different gel formats (7 cm/Ready Gel, 11 cm/Criterion, and 17 cm/
PROTEAN II Ready Gel) and stained with Bio-Safe Coomassie Stain.
2. Coomassie Brilliant Blue R-250 Stain.
a
Add enough Coomassie Brilliant Blue R-250 stain to one or more staining trays to
completely cover each gel.
b
At the conclusion of SDS-PAGE, open each gel cassette and place each gel into the
stain.
c
Place each gel on a rocker or orbital shaker and shake for at least 60 minutes.
Discard the stain when complete.
d
Destain the gels with destain solution (10% acetic acid/40% methanol in water) until
the background staining is acceptable. For best results change the destain solution
several times. The gels can be stored for several days in a solution of 10% acetic acid.
e
Figure 15 shows the expected 2-D gel images of the E. coli protein sample run on
each of the 3 different gel formats (7 cm/Ready Gel, 11 cm/Criterion, and 17 cm/
PROTEAN II Ready Gel) and stained with Coomassie Brilliant Blue R-250 stain.