(1)  Fazekas De St. Groth, S. et al. (1963) Biochim. Biophys. Acta 71, 377-391
Two new staining procedures for quantitative estimation of proteins on electrophoresis strips.
  (2)  Chrambach, A. et al. (1967) Anal. Biochem. 20, 150-154
A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis.
  (3)  Neuhoff, V. et al. (1988) Electrophoresis 9, 255-262
Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie® Briliant Blue G-250 and R-250.
  (4)  Choi, J.-K. et al. (1996) Anal. Biochem. 236, 82-84
Modified Coomassie® Blue staining of proteins in polyacrylamide gels with Bismark brown.
  (5)  Tal, M. et al. (1985) J. Biol. Chem. 260, 9976-9980
Why does Coomassie® Brilliant Blue R Interact Differently with Different Porteins?

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 approximately 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