Quantitative Analysis of Cardiac Proteins

Atrial tissue was collected and homogenized as described previously (Alvarado et al., 2017 (link)), in a buffer containing 0.9% NaCl, 10 mM Tris-HCl pH 6.8, 20 mM NaF and protease inhibitors. Equal amounts of protein, as determined by Bradford assay, were loaded. 50 µg of tissue homogenate, in Laemmli buffer, was separated by SDS-PAGE in 4–20% TGX or AnyKD precast gels (Bio-Rad). Proteins were transferred to PVDF membrane using the iblot2 transfer system (ThermoFisher) or wet transfer. Primary antibodies were as follows: anti-RyR2 (1:2000; MA3-925, ThermoFisher), SERCA2 (1:1000; MA3-919, ThermoFisher), NCX (1:1000; MA3-926, ThermoFisher), PLN (1:5000; A010-14, Badrilla), pT17-PLN (1:5000; A010-13, Badrilla), pS16-PLN (1:5000; A010-12, Badrilla), Cav1.2 (1:200; ACC-003, Alomone), GAPDH (1:10000; MAB374, Millipore). Secondary antibodies were: goat anti-mouse-HRP (1:5000; 31437, ThermoFisher) or goat anti-rabbit-HRP (1:5000; 31463, ThermoFisher). Secondary antibody concentrations were 5x higher when using the ibind Flex system. SuperSignal ECL reagent (ThermoFisher) was used to develop membranes followed by imaging with a ChemiDoc MP apparatus (Bio-Rad). Band intensities were quantified with the ImageLab software (Bio-Rad) or using ImageJ (NIH).