Bioquímica e Bioquímica Analítica

Abstrato

Rapid Detection of Proteins on One-Dimensional Polyacrylamide Gel Electrophoresis Using an Inexpensive Fluorescent Optical Brightener

Sashidhar RB, Tanuja Kosuri and Sujata Nayak

A commercial fabric fluorescent optical brightener Ranipal^® (F-OB), has been successfully employed to stain proteins on native and SDS-1D-PAGE. The F-OB was purified by using a biphasic solvent system of dichloromethane and water. The R_f value (0.63) of purified and crude F-OB were comparable on TLC. The mass spectrometry of purified F-OB indicated base peak at 414 (m/z). Absorption and emission maxima of F-OB were found to be 350 nm and 430 nm, respectively. The F-OB could stain the proteins both pre- and post-electrophoretic run, on native gel. Postelectrophoretic staining was rapid and required 20 min to visualize the stained proteins. On the other hand, in SDS gels, an additional 20 min was required for the extraction of SDS before staining the proteins with F-OB. SDS was found to interfere with binding of F-OB to proteins. Varying concentrations of molecular weight markers were loaded and their fluorescent intensity was plotted against the concentration of the proteins. The r² values ranged from 0.965 to 0.997 indicating excellent linearity. The detection for carbonic anhydrase was in the range of 8.0-800 ng. Unlike most of the dyes used for protein staining, staining with F-OB could be carried out in tank buffer (2 mg/100 mL) and was also reversible. The F-OB, perhaps would be the most cost-effective fluorescent dye to stain the proteins (US $ 0.04/25.0 g). The F-OB was found to be simple, safe, sensitive, less time consuming and economical fluorescent dye as an alternative, for staining proteins on polyacrylamide gels.