Protein a agarose beads

Frozen mesenteric artery was thawed, homogenated in 1 ml cell lysate, and centrifuged at 10000 g for 15 min. The concentration of protein extracts was measured by the BCA protein Assay Kit (Pierce, USA). Protein extracts were incubated with normal rabbit IgG (50ul) in an ice bath for 1 h, precleared with proteinA-agarose beads (Bio-Rad, Germany) at 4℃ for 30 min and centrifuged at 12000g for 10 min at 4℃. Then the 80 μl of the supernatant (about 50 μg protein) were incubated with α1 adrenergic receptor antibody at 4℃ overnight, washed in lysis buffer and centrifugated at 1000g for 5 min at 4℃. The precipitates were then stored in -80℃ or detected by Western blot with the β-arrestin-2 antibody.

Cells were lysed at 4°C in pre-cold radioimmunoprecipitation assay lysis buffer (9806; Cell Signaling Technology) and cell lysates were cleared by brief centrifugation (12,000 × g for 10 minutes).⁸⁷ (link) Concentrations of proteins in the supernatant were determined by bicinchoninic acid assay (23225; Thermo Fisher Scientific). Before immunoprecipitation, samples containing equal amounts of proteins were precleared with protein A agarose beads (9863; Cell Signaling Technology) at 4°C for 3 hours and subsequently incubated with irrelevant IgG or specific antibodies (5 μg/mL) in the presence of protein A agarose beads overnight at 4°C with gentle shaking. After incubation, protein A agarose beads were washed extensively with phosphate-buffered saline and proteins were eluted by boiling in 2 × Laemmli sample buffer (161-0737; Bio-Rad) before sodium dodecyl sulfate–polyacrylamide gel electrophoresis.