Efficient and modified protocol for zymography to detect muscle specific calpain activity

•Calpain plays critical roles in diseases like atrophy/cachexia/sarcopenia as a result it acts as direct therapeutic target.•Casein zymography is the only technique for detecting active calpain activity.•Inefficiency of reported protocols for calpain in muscle samples is a major limitation.•Presenting modified version of protocols for calpain detection for all kind of muscle samples.

Calpain, a proteolytic enzyme plays critical role in the skeletal muscle physiology by maintaining the protein metabolism. Up-regulation in the activity of such enzyme under diverse clinical settings (i.e. diabetes, cancer, AIDS, chronic heart failure, immobilization, aging etc) leads to loss in muscle proteins and causes atrophy/cachexia/sarcopenia. Beyond a reduced survival rate, atrophy is also linked to poor functional status and quality of life. Thus, easy detection of calpain at very early stage is highly desired under such settings so that specific therapy or antagonist could be given in time. Multiple methods are available (zymography, qPCR and immunoblotting) and among these zymography is the only approach which actually detect calpain activity. In the present manuscript, we have improvised two zymography protocols which are able to detect calpain in all muscle cells and tissues. Result shows that modified protocols can detect calpain activity even at low proteins concentration (<10 μg) in muscle cells (C2C12 myoblasts, proliferating state; myotubes, differentiating state) and tissues (cardiac and skeletal) in a single gel in comparatively short time span. Overall, purpose of the present study is to provide relatively simple and well described experimental protocol which can be used for muscle-specific calpain study.

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