Quantifying Myosin and Actin Content

Determination of myosin and actin content were performed as previously described by our laboratory [19 (link)] and others [25 (link)]. Briefly, SDS-PAGE preps were made using 10 μL resuspended myofibrils, 65 μL distilled water (diH₂O), and 25 μL 4x Laemmli buffer. Samples (15 μL) were then loaded on pre-casted gradient (4–15%) SDS-polyacrylamide gels (Bio-Rad Laboratories) and subjected to electrophoresis at 200 V for 40 minutes using pre-made 1x SDS-PAGE running buffer (Ameresco). Following electrophoresis gels were rinsed in diH₂O for 15 minutes, and immersed in Coomassie stain (LabSafe GEL Blue; G-Biosciences; St. Louis, MO, USA) for 2 hours. Thereafter, gels were destained in diH₂O for 60 minutes, bright field imaged using a gel documentation system (UVP), and band densities were determined using associated software. Given that a standardized volume from all samples were loaded onto gels, myosin and actin band densities were normalized to input muscle weights for relative expression. Our laboratory has reported that this method yields exceptional sensitivity in detecting 5–25% increases in actin and myosin content [19 (link)]. Notably, actin and myosin content were the only two myofibrillar protein targets of interest given that the combination of these proteins make up a majority (~70%) of the myofibrillar protein pool [20 (link)].