Coomassie Brilliant Blue
 
Proteins are most frequently detected after gel electrophoresis by fixing them in the gel, so that they do not diffuse further, and then staining them to produce a colored zone. The most sensitive stain is silver stain, but the stain used most frequently is Coomassie Brilliant Blue. It has two frequently encountered forms, R250 and G250, whose structures are depicted in Figure 1. Although the two dyes are structurally very similar, they require different physical and staining procedures and are not interchangeable in any particular protocol. These two dyes do not react chemically with proteins but merely form noncovalent complexes. The interaction with proteins is believed to be primarily ionic and involves the acidic sulfonate groups on the dye and basic groups on the protein, but nonpolar van der Waals forces are probably also involved. Consequently, the dyes do not bind equally to all proteins, so two electrophoretic bands with the same blue color need not contain the same amount of protein (1). 
Figure 1. The chemical structures of the R250 and G250 forms of Coomassie Brilliant Blue. They differ only in the nature of the group R.
Electrophoresis gels are readily stained by placing the gel in a liquid containing the Coomassie dye plus an agent to fix the protein bands, usually acetic acid plus methanol; a solution of 10% (w/v) trichloroacetic acid plus 10%(w/v) sulfosalicylic acid is very effective for fixing and staining. Fixed and insoluble proteins bind the dye tightly. It is usually necessary to remove the excess dye from the staining mixture before the bands on the gel can be seen. This is usually accomplished simply by washing the gel in the fixative solution, but it can also be accomplished by adding an agent, such as polyurethane foam, that binds the excess dye tightly. It is important not to remove all of the excess dye from the solution because then the dye bound to the protein bands dissociates and the gel becomes bleached. The procedure is simplified if the Coomassie dye is only slightly soluble in the original staining mixture; the protein takes up the dye from the solution, but the background color remains weak.
A number of other dyes, such as Ponceau S and Amido black, can be used in the same way, but they are not as sensitive as the Coomassie dyes. Coomassie Brilliant Blue detects approximately 0.1 µg of protein in a band on a polyacrylamide gel.
Coomassie Brilliant Blue, particularly G250, is also used to quantify the amount of protein in solution, which is known as the Bradford assay (2). The dye complexed to protein has an altered absorbance spectrum. Under certain acidic conditions, the absorbance maximum shifts from 465 to 595 nm upon binding to a protein. Even though different proteins give somewhat different responses in this assay, its simplicity makes it widely used.
References
1. M. Tal, A. Silberstein, and E. Nusser (1980) J. Biol. Chem. 260, 9976–9980.
2. M. M. Bradford (1976) Anal. Biochem. 72, 248–254.