Hrp conjugated goat anti rabbit igg

Whole cell lysate protein concentrations were measured by Bradford assay to ensure equal protein loading. Proteins were separated by polyacrylamide gel electrophoresis using 4–20% gradient gels. Proteins were then transferred to 0.2 µm pore size polyvinylidene fluoride membranes. The membranes were blocked in 5% BSA in tris-buffered saline buffer containing Tween 20 (TBST) for 1.5 hours. The membranes were then incubated in primary antibody solutions containing 1% BSA in TBST overnight at 4 °C. Following primary antibody incubation, the membranes were washed 3 times for 10 minute each wash in TBST. After washing, the membranes were incubated in secondary antibody solutions containing 1% BSA in TBST for 1 hour. SuperSignal West Pico chemiluminescence substrate was used for detection. Images were recorded on an Azure C300 system using automatic exposure settings to prevent oversaturation of bands. The following primary and secondary antibodies were used: mouse anti-GAPDH (1:20,000; Novus Biologicals), mouse-anti-Myosin-hc (1:250; Novus Biologicals), rabbit-anti-parvalbumin (1:10,000; Novus Biologicals), horseradish peroxidase (HRP)-conjugated goat-anti-mouse IgG (1:1000; Novus Biologicals) and HRP-conjugated goat-anti-rabbit IgG (1:1000; Novus Biologicals). Densitometry was performed in ImageJ using the Gel Analysis tool.