A new staining procedure for proteins in polyacrylamide gels has been developed. This procedure, utilizing Coomassie brilliant blue G250, is rapid (as quick as 30 min) and simple to perform, requires little or no destaining, and has a sensitivity of a least 1 pg of protein per band. It can be used to stain proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by polyacrylamide gel electrophoresis in nondissociating solvents.
Our staining procedure is based on the procedures developed by Diezel et al. ( 1) and Malik and Berrie (2). Diezel et al. ( 1) introduced the use of Coomassie brilliant blue G250, a methyl-substituted triphenylmethane dye. This dye, when used in 12.5% trichloroacetic acid, does not effectively stain polyacrylamide gels, and, thus, the undesirable stained gel backgrounds are not obtained. The interior portions of the protein bands, however, are usually not stained. If, after treatment with this dye, the gels are stored in 5% acetic acid, an intensification of the stained protein bands occurs as the protein-bound dye becomes soluble, diffuses into the gel, and binds again to the interior portions of the protein bands. In our hands, the procedure of Diezel et al. (1) was significantly less sensitive than the conventional procedure using Coomassie brilliant blue R250 (3).
Malik and Berrie (2) described a protein-staining procedure with Coomassie brilliant blue R250 prepared using sulfuric acid, KOH, and trichloroacetic acid. With this procedure, rapid staining of the protein bands occurs, but, in our hands, the gel background became significantly stained, and a destaining step with 0.2% (v/v) H&SO, was required to reduce the background. The destaining caused a significant reduction in the intensity of the protein-stained bands. Our staining procedure combines features of both the Diezel et al. (1) and the Malik and Berrie (2) procedures. Stain preparation. Coomassie brilliant blue G250 (xylene brilliant cyanin G) was purchased from K and K Laboratories, Inc. (Catalog No. 27551). To a 0.2% (w/v) aqueous solution of the dye was added an equal volume of 2 N H,SO,. After the solution was mixed well, it was allowed to 580 Copyright