Modified Zymography Protocol: A Rapid and Sensitive Method for Monitoring Calpain Activity

Calpain is a key proteolytic enzyme involved in muscle development. Overactivation in diseases can lead to muscle atrophy, and its role in postmortem meat tenderization affects consumer acceptability. To develop a rapid and sensitive zymography protocol for the efficient detection of calpain activity in skeletal muscle cells, we addressed the limitations of traditional methods that require lengthy assay times and high protein inputs. A previously published zymography method was modified by increasing the Ca²⁺ concentration from 4 to 8 mM and reducing the incubation time from overnight to 6 h. Calpain activity was assessed in differentiated C2C12 myotubes using varying protein concentrations (1-8 μg), and band intensity was quantified using ImageJ software. The optimized conditions enhanced the detection sensitivity up to tenfold and enabled reliable visualization of calpain activity using only 1 μg of protein within 8 h. The 8 mM Ca²⁺ incubation buffer yielded stronger, clearer lytic bands compared with 4 mM, indicating improved calpain activation. This modified zymography protocol significantly reduces assay time and protein requirement while maintaining high sensitivity. The method serves as an efficient tool for studying calpain-mediated proteolysis in muscle atrophy research and postmortem meat quality evaluation.