Quantitation of proteins on Coomassie blue-stained polyacrylamide gels based on spectrophotometric determination of electroeluted dye.
A procedure for quantitating proteins on Coomassie blue-stained polyacrylamide gels is presented. The method is based on the observations that the dye is rapidly eluted electrophoretically from stained protein bands or spots in the presence of sodium dodecyl sulfate, and that the eluted dye is nondialyzable. Protein may therefore be assayed indirectly by measuring the dye in electroeluents spectrophotometrically. Moreover, the stain elutes more rapidly than the protein, allowing separate recovery of the protein for further analysis. The assay is independent of band or spot size, and does not involve physical disruption of the gel piece or any chemical treatment harsher than the staining process itself. The technique has been applied to the contractile proteins myosin, actin, and commercially obtained standards resolved by one-dimensional electrophoresis, and to proteins in nuclear extracts of HeLa cells on two-dimensional gels.
D000818:Animals; D002642:Chick Embryo; D004396:Coloring Agents; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007201:Indicator Dilution Techniques; D011506:Proteins / Q000032:analysis*; D012394:Rosaniline Dyes; D013053:Spectrophotometry; D013194:Staining and Labeling
D004396:Coloring Agents; D011506:Proteins; D012394:Rosaniline Dyes; C024757:Coomassie brilliant blue R