An immunological method to assess protein degradation in post-mortem muscle

A method for determining proteolysis of any specific protein in muscle is demonstrated. The protocol involves the preparation of a specific antibody which is used in immunoblotting total protein extracts from post-mortem muscle. In the present study an antibody to bovine myosin heavy chain was prepared that reacts with intact myosin heavy chain as well as proteolytic fragments that are generated by proteases. We show that little, if any, myosin heavy chain fragments are generated during prolonged aging of muscle at 4°C. In contrast, storage of muscle at 37°C results in the rapid breakdwon of myosin heavy chain to immunologically detectable peptides. Using a monoclonal antibody to titin, we demonstrate that this protein is degraded at 4°C during the aging period, and that, between 2 and 3 weeks following slaughter, no undergraded titin is detectable. This method is suitable for the analysis of any protein that can be separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE).