Tissue samples are lysed in buffer supplemented with leupeptin to maintain enzymatic activity. The samples are standardized for protein concentration and then loaded into a polyacrylamide gel for SDS-PAGE. After separation of proteins by molecular weight is complete, the gel is incubated in a renaturing buffer to restore enzymatic activity. During loading, a non-reducing buffer was used to preserve protein disulfide bonds. After the renaturing period, the gel is then incubated in assay buffer to allow the now active cathepsins to proteolyze the gelatin impregnated in the gel. This process takes place overnight. The next day, the gel is analyzed for regions of gelatin degradation by coomassie blue staining. Patches of gel that are no longer blue following a destain wash are noted. These bands are then correlated to their respective molecular weights in order to identify which cathepsins were active in the sample.
Cathepsin K detection by zymography