Goat anti rabbit igg horse radish peroxidase secondary antibody

HEK293 cells or neonatal mouse cardiomyocytes were washed with PBS and solubilized using a lysis buffer containing 5 mmol/L EDTA, 50 mmol/L Tris‐HCl (pH 7.4), 150 mmol/L NaCl, 10% glycerol, (Sigma‐Aldrich), 1% Nonidet P‐40 (Thermo Scientific), and Complete mini protease inhibitors (Roche). Whole‐cell lysates were then collected and centrifuged, and protein quantification was performed using the DC Protein Assay Kit II (Bio‐Rad Laboratories). Lysates were prepared and loaded on 7% SDS‐polyacrylamide gels and then transferred to nitrocellulose membranes, as described previously.^{21 (link)} Nonspecific binding sites were blocked using 5% nonfat dry milk and 0.1% Tween‐20 in PBS. Membranes were probed with a rabbit anti‐ERG primary antibody (1:10 000) containing a C‐terminal ERG epitope^{5 (link)} and then washed with 0.1% Tween‐20 in PBS to remove excess antibody. Membranes were probed with a goat anti‐rabbit IgG horse radish peroxidase secondary antibody (1:10 000; GE Healthcare Biosciences). Protein bands were visualized using ECL Western blotting detection reagents (GE Healthcare Biosciences).