Rabbit trueblot anti rabbit igg hrp

Cells were lysed using ELB buffer (250mM NaCl, 50mM Hepes pH 7.0, 5mM EDTA and 0.1% NP40), and protein samples were pre-cleared using TrueBlot anti-Rabbit IgG Magnetic beads (Rockland p/n 00–1800-50). Samples were then incubated with 2μg of antibody targeting EYA2: anti-EYA2 IgG (Sigma, HPA027024) and 50μl of Trueblot magnetic beads overnight at 4 °C, while gently rocking. The following day, beads were washed using TBS, and proteins were dissociated from the beads by boiling the sample with loading buffer prior to the Western Blotting analyses. Of note, secondary antibodies used during Western Blot analyses only recognize non-denatured IgG (Rabbit Trueblot Anti-Rabbit IgG HRP, Rockland, p/n 18–8818-31) and Mouse Trueblot ULTRA Anti-Mouse IgG HRP, Rockland, p/n 18–8817-33).

Confluent 60 mM plates of approximately 1 million neonatal myocytes were washed twice with PBS and lysed with 1 ml HSE buffer (20 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 5 mmol/L EDTA, 1% Triton X-100) supplemented with protease inhibitors (AEBSF, benzamidine, leupeptin/pepstatin) and okadaic acid (500 nmol/L). Following centrifugation at 13.2 krpm at 4 °C, soluble lysates were incubated with 5 μL mAKAP or nesprin antibodies in the presence of 25 μLTrueBlot Anti-Rabbit Ig agarose Beads (Rockland) overnight at 4 °C. Beads were pelleted and washed three times with HSE buffer and boiled in 25 μL 2× SDS loading buffer. Samples were size-fractionated by 5% (for RyR2) or 7.5% SDS-PAGE and transferred to nitrocellulose membranes. Blots were blocked in 5% milk for one hour, followed by incubation in primary antibody overnight at 4 °C. Following washes, membranes were incubated with secondary antibodies (Rabbit Trueblot Anti-Rabbit IgG HRP, Rockland, 1:5000) for one hour. Signals were visualized with an enhanced chemiluminescence reagent (Abcam) and exposed to X-ray film. Quantification was determined by ImageJ analysis.