Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) with Activity Staining for Polyphenol Oxidase...

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a popular technique for the separation and characterization of proteins. Whereas most proteins are denatured by the anionic detergent SDS, the enzyme polyphenol oxidase (PPO) is activated by low SDS concentrations, provided that the samples are not heated and reduced. Direct activity staining for PPO is therefore possible on SDS-PAGE gels. Color development from ortho-quinone reaction products can be enhanced with coupling reagents such as 4-amino-N,N-diethylaniline (ADA). As demonstrated with Agaricus bisporus (white and brown button mushroom) extracts, multiple PPO forms can be clearly resolved on SDS-PAGE gels. The method described here encompasses sample preparation, electrophoresis, and activity staining. It is a rapid, simple, and high-resolution technique that enables the comparison of PPO forms across several samples without the need for protein purification.