Cost-Effective Method for the Detection of Lipases and Proteases in Crude Extracts of Microbial Proteins Using Gel Zymography.

The transfer zymography technique detects and determines the molecular mass of enzymes in crude microbial protein extracts. Proteins are first resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then renatured by removing SDS with Triton X-100. This technique allows proteins to be excised from the gel for further analysis and identification via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Transfer zymography uses a separate gel copolymerized with a substrate, avoiding substrate interference and providing accurate mass determination. Electrotransfer is the preferred method because of its speed. The resolving gel is incubated in buffer, leaving the protein negatively charged; then, the proteins are electrotransferred to a casting gel with the substrate. If the transfer buffer is not suitable, capillary transfer is used. This method does not require the protein to be negatively charged. The renatured SDS-PAGE gel is placed against the copolymerized gel in a sandwich assembly, allowing the buffer to transfer enzymes by capillary action. Both methods visualize active protease or lipase regions as clear bands on the casting gel. This chapter details a protocol for analyzing enzymes in microbial protein extracts using electrotransfer for protease detection and capillary transfer for lipase detection. This methodology was applied to the entomopathogenic fungus Beauveria pseudobassiana RGM 2184.