Immunoblotting Sample Clauses

Immunoblotting. 1. Following the focusing step, remove the gel from the chamber floor, and place it on a Blotter D with sample number one on the left. Remove and discard the electrode xxxxx and the blotters.

Immunoblotting. The immunoblotting methods were performed as described in previous studies (Xxxxxxxx et al., 2006; Xxx et al., 2008; Xxxxxx et al., 2000). Briefly, fresh tissue samples were homogenized in buffer H (210 mM mannitol, 70 mM sucrose, 1 mM EGTA, 5 mM HEPES) with protease inhibitors (1 mg/ml apoprotinin, leupeptin, and pepstatin A) and assayed for protein concentration using Bio-Rad protein assay (BioRad, Hercules, CA) according to manufacturer's protocol. The cell lysate was obtained from cultured cells that were grown on 100 mm plates, washed two times in PBS, pH 7.4, and incubated in 400 µl of cell lysis buffer (Promega, Madison, WI) containing protease inhibitors (Roche, Mannheim, Germany), 700 U/ml DNAse I (Gibco, MD), and 1% β-mercaptoethanol (Sigma, St. Louis, MO) for 15 min at room temperature. Cells were scraped and lysate was centrifuged at 10,000×g for 10 min. Supernatant was collected as the soluble fraction. Protein content was assayed using Bio-Rad protein assay (BioRad, Hercules, CA) according to manufacturer’s protocol. Tissue samples were prepared at 4°C. The fresh tissue was completely homogenized with a sonicator in an ice-cold buffer solution containing protease inhibitor cocktail (1 tablet per 50 ml; Roche Diagnostics GmbH, Mannheim, Germany), 1.0% Triton X-100, 20 mM HEPES, 10 mM EDTA, and 2 mM Na3VO4. The homogenate was then centrifuged for 5 minutes at 2,000 rpm to remove tissue debris. The lysates were eluted with 6× SDS-PAGE sample buffer. Samples were loaded on a 12% acrylamide mini gel and run at 130 V in running buffer (0.1% XXX, 000 mM Tris base, 1 M glycine) on a Bio-Rad minigel apparatus. Gels were rinsed in transfer buffer (10% methanol, 25 mM Tris, 192 mM glycine) for 10 min, apposed to polyvinylidene difluoride (PVDF) membrane (Millipore, Billercia, MA U.S.A.), and transferred overnight at 35 V under cooling conditions. PVDF membranes were incubated in KPL blocker for 3 h on a shaker at room temperature and then incubated in blocker containing primary antibody raised against mGluR8 (1:750) overnight at 4°C on a shaker. Membranes were thoroughly rinsed in PBS/Tween, incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, and rinsed thoroughly in PBS/Tween. The blots were detected using Super-signal West Dura extended duration substrate (Pierce, Rochford, IL). The blots were developed using Xxxxxxx Kodak (Rochester, NY).