Systematic optimization of multiplex zymography protocol to detect active cathepsins K L S and V in healthy and diseased tissue: compromise between limits of detection reduced time and resources

摘要

Cysteine cathepsins are a family of proteases identified in cancer, atherosclerosis, osteoporosis, arthritis and a number of other diseases. As this number continues to rise, so does the need for low cost, broad use quantitative assays to detect their activity and can be translated to the clinic in the hospital or in low resource settings. Multiplex cathepsin zymography is one such assay that detects subnanomolar levels of active cathepsins K, L, S, and V in cell or tissue preparations observed as cleared bands of proteolytic activity after gelatin substrate SDS-PAGE with conditions optimal for cathepsin renaturing and activity. Densitometric analysis of the zymogram provides quantitative information from this low cost assay. After systematic modifications to optimize cathepsin zymography, we describe reduced electrophoresis time from 2 hours to 10 minutes, incubation assay time from overnight to 4 hours, and reduced minimal tissue protein necessary while maintaining sensitive detection limits; an evaluation of the pros and cons of each modification is also included. We further describe image acquisition by smartphone camera, export to Matlab, and densitometric analysis code to quantify and report cathepsin activity, adding portability and replacing large scale, darkbox imaging equipment that could be cost prohibitive in limited resource settings.