Titin Reactivity Analysis by SDS-PAGE

To evaluate titin reactivity for quality control and experimental success, fiber samples treated with or without TEV protease were analyzed by agarose-strengthened 1.8–2.4% SDS–PAGE and visualized with Coomassie blue, as previously done (Prado et al., 2005 (link); Unger et al., 2017 (link)). Relative band intensities between intact and cut N2A titin were used to measure % of total titins cut. Western blotting was performed as described elsewhere (Unger et al., 2017 (link)). To detect HaloTag-TEV titin, we used the primary antibody (anti-HaloTag pAB, Promega 6928) and secondary antibody (anti-rabbit HRP, Acris, Herford, Germany, R1364HRP). Signals from HRP‐conjugated secondary antibodies were visualized by chemiluminescence (Amersham ECL start Western blotting detection reagent, GE Healthcare) and recorded using the ImageQuant LAS 4000 Imaging System (GE Healthcare). Signal intensity was quantified by densitometry using the ImageQuant TL software (GE Healthcare). A subset of TEV-treated fibers (n = 5/genotype) were also loaded onto Coomassie-stained, agarose-strengthened, 2.4% SDS–PAGE, titin gels to measure the MyHC:titin, titin:NEB, and MyHC:NEB protein ratios after active contraction.