Protein g magnetic surebeads

Immunoprecipitation assays were performed by transfecting a bait GFP fusion protein into HeLa cells as discussed above. Approximately eight million cells were harvested for each sample and lysed into Pierce lysis buffer (Thermo) containing Complete protease inhibitors (Roche). Lysate was clarified by centrifugation at 10,000 × g for 30 min. Pellet and a portion of supernatant were reserved as input. The remaining lysate was incubated with 4 µg of goat anti-GFP overnight, followed by immunoprecipitation with Protein G Magnetic SureBeads (BioRad). Beads were washed three times in Pierce lysis buffer and eluted into NuPAGE Sample Buffer (Invitrogen). The resulting eluent was run onto a NuPAGE 4–12%, Bis-Tris gel (Thermo), transferred to nitrocellulose and probed with primary antibody to be visualized with horseradish peroxidase conjugated secondary antibody. The antibodies used were NOLC1 antibody (NovusBio NBP1-2298), GFP antibody (Invitrogen A-11122), and anti-rabbit IgG Horseradish Peroxidase-Linked Species-Specific Whole Antibody (GE HealthCare NA934).

The rat hepatocyte lysates (prepared as for immunoblots) were precleared with Protein G Magnetic SureBeads (Bio-Rad Laboratories, Hercules, CA). The protein (50 μg) was immunoprecipitated by adding antibodies specific to Keap1 (Santa Cruz, CA, USA) or normal rabbit IgG and rotated for 15 min at room temperature. Immune complexes were then precipitated with protein G SureBeads. The bound proteins were then eluted by incubation with 4× SDS-PAGE sample buffer for 30 min at 37°C and analyzed by Western blotting with an anti-Nrf2 antibody (Abcam, Cambridge, MA, USA).

Cells were grown in 10 cm dishes were resuspended in ice cold non-denaturing immunoprecipitation buffer containing 20 mM Tris HCL, 137 mM NaCl, 1% NP-40, 2 mM EDTA, and protease inhibitors (Roche). Total cell protein was normalized using a protein assay (BioRad) and 1 mg of protein from each sample was incubated with 2 µg of capture antibody targeted against GRP78 (Santa Cruz Biotechnology; SC-1050) and rotated on a platform for 24 h at 4 ˚C. Following incubation, samples were exposed to 100 µl of Protein G magnetic Surebeads (BioRad) for an additional 2 h on a rotating platform at 4 ˚C. Beads conjugated to the anti-GRP78 antibody were subsequently isolated and the remaining sample was collected and labeled “input” for use as controls. The magnetic beads underwent four consecutive washes using the non-denaturing IP buffer and resuspended and boiled in 100 µl of 4X SDS-PAGE sample buffer.