Identification of ELC interacting proteins in zebrafish heart

The essential myosin light chain (ELC) is part of the cardiac myosin motor regulating the contractility of the heart. Recently, we have demonstrated ELC phosphorylation to be responsible not only for basal maintenance of cardiac contractility, but also for the adaptation of sarcomere function to increased demands in adult zebrafish (Scheid et al. Cardiovascular Research, 2016). However, the direct interacting kinases, phosphatases as well as the signaling pathway mediating the ELC phosphorylation are mostly unknown. Here, we aim to identify ELC interacting proteins in zebrafish heart tissue using an ELC-specific precipitation assay followed by mass spectrometry. In brief, ELC specific co-immunoprecipitation was performed by using the Dynabeads Co-immunoprecipitation Kit (Invitrogen, #14321D). Custom-specific polyclonal ELC antibodies (Eurogentec, peptide for immunization: NH2-CAPVPETPKEPEVDLK-CONH2) against zebrafish ELC were covalently coupled to DynabeadsTM M-270 Epoxy (Invitrogen, #14302D) and whole zebrafish heart protein was used as input. All steps were carried out according to the manufacturer's instructions.