Abstract
AbstractA systematic analysis of protein staining in polyacrylamide gels with Coomassie Brilliant Blue (CBB) R‐250 and G‐250 using a high resolution densitometer allowing for quantitative measurements during staining and destaining has revealed that none of the published procedures allows quantitative measurements. Protein staining with CBB R‐250 in methanol/water/acetic acid is poor, as is staining with CBB G‐250 in trichloroacetic acid or perchloric acid, the latter two, however, allowing for a weak background staining. Consequently using the colloidal properties of the CBB dyes, stronger for G‐250 than for R‐250, it is possible to increase the sensitivity of protein staining to a detection limit of 0.7 ng bovine serum albumin/mm2 gel. In addition, sensitive protein staining on a clear background is possible. Recipes are described (Section 3.11) for intensified protein staining with CBB G‐250 using trichloroacetic acid or perchloric acid on a clear background. Optimal staining of proteins on a clear background can be performed with phosphoric acid and CBB G‐250 in the presence of ammonium sulfate since under these conditions the colloidal state of the dye is optimized. Furthermore, conditions are described which allow the stable fixation of the protein‐dye complex. Combining the optimized staining conditions with the stable fixation in 20% ammonium sulfate allows for stepwise staining for e. g. detection of weak spots in addition to intense protein spots. The dependence of different staining procedures on gel thickness, gel concentration and compounds routinely used in polyacrylamide gel electrophoresis is also analysed. Calibration curves and application of the new procedure to biological material demonstrate its wide applicability. Convincing arguments for the colloidal properties of the CBB dyes are presented, formulating the rationale for intensified protein staining with CBB dyes in polyacrylamide gels without background staining.
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