Coomassie® Brilliant Blue R-250 is one of the most commonly used stains for proteins, after their separation by polyacrylamide gel electrophoresis. The protein-dye complex has an absorption maximum at 549 nm, the dye without protein at 555 nm （in 0.01 M citrate buffer, pH 3）. The intensity in staining of proteins probably depends on the basicity of a protein （5）. Per positively charged amino acid approximay 1.5 - 3 molecules of Coomassie® will be bound. This variation complicate the exact protein determination with albumin as a standard, since this protein contains more basic amino acids than many other proteins （5）.
There do exist many protocols for sensitive staining procedures with Coomassie® （e. g. ref. 3, 4）. The sensitivity reaches a limit at 25 ng protein （4）. We recommend the following protocol:
I. Staining solution: 0.1 % Coomassie® Brilliant Blue R-250 （Prod.-No. A1092）
20 % methanol （or ethanol）
10 % acetic acid
The SDS gel （without 'stacking gel'） is stained for 1 hour at 60°C or for 2 hours at 50°C or over night at RT.
II. Destaining solution: 20 % methanol （or ethanol）
10 % acetic acid
Destain the gel for 3 - 4 hours at 50 - 60°C. Add some sponges.
Subsequently wash the gel for 15 minuts in water and dry under vacuum at 60°C for 2 - 3 hours.