Biomedical Engineering Reference
In-Depth Information
TCA, equilibrate the gel twice for 20 min in Soln. B prior to staining.
Destaining has to be done alternating with Soln. B and B
.
The destained gel may be stored in Soln. B for several days, but
weak bands fade slowly.
Densitometry of stained gel should be done at 590 nm.
By modification of Soln. A (0.05 g of dye are dissolved in 10 ml
Variant for thin gels
glacial acetic acid and filled up with deionized water to 100 ml),
staining is done at 60
◦
C and destaining is done with 10% acetic
acid in deionized water.
2.4.1.3 Coomassie Brilliant Blue R250 Combined
with Bismarck Brown R
Gels are stained very sensitive and quick by combination of
Coomassie Brilliant Blue R250 with Bismarck Brown R (Basic
Brown 4; C.I. 21010). In our opinion, this is the best staining method
using organic dyes.
A .2g Coomassie Brilliant Blue R250 are dissolved in 40 ml
Solutions/Reagents
methanol or non-denaturated ethanol; then 10 ml glacial
acetic acid are added and the solution is filled up to 100 ml
with deionized water. Stable at RT
B . 5g Bismarck Brown R are dissolved in 40 ml methanol or
non-denaturated ethanol; then 10 ml glacial acetic acid are
addedandthesolutionisfilledupto100ml with deionized
water. Stable at RT.
C1vol. A is mixed with 0.75 vol. B immediately prior use.
D
destaining solution
: 10% acetic acid (v/v), 30% methanol or
non-denaturated ethanol or isopropanol (v/v) in deionized
water
Thegelisfixedfor5-10min in Soln. D after electrophoresis and
stained with Soln. C (about 20 ml for a 8
0.1 cm gel) for 20 min
with agitation. Flocculation of dye does not influence staining.
Destaining is done with Soln. D.
Sensitivity is about twice the standard protocol without Bis-
marck Brown.
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6
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References
Choi J-K, Yoon S-H, Hong H-Y, Choi D-K, Yoo G-S (1996) Anal Biochem
236:82
2.4.1.4 Fast Green FCF
(Food Green 3; C.I. 42053)
A . 5g Fast Green are dissolved in 30 ml ethanol and 10 ml
Solutions/Reagents
glacial acetic and filled up to 100 ml with deionized water
B
destaining solution
: 10% acetic acid (v/v), 30% methanol or
denaturated ethanol (v/v) in deionized water
