A Method to Exchange Recombinant Differentially Phosphorylated Rhodamine-Labeled Cardiac RLC into Permeabilized Cardiac Trabeculae

Electron tomography of muscle cross- bridge by regulatory light chain labelling with APEX2

“…In rabbit skeletal muscle the myosin cross-bridges are distributed at a fixed interval of 13.5 nm (Haselgrove, 1980). Based on Toepfer et al 0.5 mg/ml recombinant protein would provide 50 % of native RLC replacement with recombinant RLC (Toepfer et al, 2012). If our sample incorporates 50 % exchange, then we should able to visualise crossbridges periodicities due to the presence of RLC-APEX2.…”

“…used a well-established TFP based protocol. Over the past decade our lab regularly achieved 80% exchange efficiency for ELC and RLC by employing the TFP-based protocol (Toepfer et al, 2012). The exchange efficiency was determined by comparing the fluorescence intensity of rhodamine labelled light chain to non-exchanged muscle.…”

“…The exchange efficiency was determined by comparing the fluorescence intensity of rhodamine labelled light chain to non-exchanged muscle. Moreover, the exchanged muscle showed no physiological changes compared to the native muscle based on isometric force analysis (Caorsi et al, 2011, Toepfer et al, 2012.…”