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Title: Technical note: Protocol for electrophoretic separation of bovine myosin heavy chain isoforms and comparison to immunohistochemistry analysis. Author: Scheffler TL, Leitner MB, Wright SA. Journal: J Anim Sci; 2018 Sep 29; 96(10):4306-4312. PubMed ID: 30032293. Abstract: Myosin heavy chain (MyHC) isoform composition is a primary determinant of contractile speed of muscle fibers. Currently, bovine MyHC isoforms are evaluated using time-consuming histochemical analysis by immunflourescence or ATPase activity. Electrophoretic separation of MyHC isoforms is more rapid; however, a reliable procedure without use of gradients has not been validated for cattle. Therefore, our objectives were to develop and validate a procedure for separating bovine MyHC isoforms (I, IIa, and IIx) using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and compare results to immunohistochemistry (IHC) analysis. Muscle samples were collected from masseter, sternomandibularis, diaphragm, longissimus lumborum, and cutaneous trunci within 1.5 h postmortem. To determine appropriate conditions for electrophoretic separation, several parameters of gel composition were varied. Bovine MyHC isoforms were clearly separated by increasing glycerol content of polyacrylamide gels to 37%. Identity of MyHC isoforms was confirmed using western blotting. Percent MyHC composition evaluated by gel electrophoresis was consistent with IHC (P > 0.2). Thus, SDS-PAGE produces clear separation of MyHC isoforms, and is a viable alternative to IHC-based methods.[Abstract] [Full Text] [Related] [New Search]