Crescendo western hrp substrate

Whole-cell lysates were isolated using radioimmunoprecipitation (RIPA) buffer supplemented with protease inhibitor (Sigma), phosphatase inhibitor (Sigma), and phenylmethane-sulfonylfluoride (PMSF) (Sigma) as previously described (15 (link)). Protein concentrations were determined using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific Inc.), and samples were separated by electrophoresis on sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels. The Precision Plus Protein Kaleidoscope molecular weight marker (Bio-Rad, Hercules, CA) was utilized to confirm expected size of target proteins. Gel transfer to blotting membrane was completed using Trans-Blot Turbo Transfer System (Bio-Rad) per manufacturer’s protocol. Antibodies were utilized according to the manufacturers’ recommendations. Immunoblots were developed with Immobilon Classico or Crescendo Western HRP Substrate (EMD Millipore, Millipore Sigma, Burlington, MA). Blots were stripped with stripping solution (Bio-Rad) at 65°C for 20 minutes and then re-probed with selected antibodies. Equal protein loading between samples was confirmed using β-actin as an internal control.

One half of an 80% confluent 15-cm dish of transgenic MEFs was lysed in 100 μL RIPA buffer plus protease and phosphatase inhibitors. Samples were incubated on ice for 10 minutes and centrifuged at 13,000×g for ten minutes at 4°3. Samples were normalized to 5 μg/μL and ran on a 12% tris-glycine gel. Approximately 20 μg of protein was loaded in to each well. Gels were transferred in transfer buffer with 20% methanol at 90V for 60 minutes at 4°C to a PVDF membrane activated with methanol. After transfer, membranes were washed once with water, rinsed in methanol for one minute, and dried at 37°3. Membranes were incubated with antibodies at 1:1000 dilution overnight at 4°C unless otherwise specified. Antibodies used are: SOD2 (Enzo sciences 1:5,000), OXPHOS Cocktail (Abcam), mt-COX3 (Santa Cruz), GAPDH (Life Technologies, 1:10,000), HSP60 (Cell Signaling), Actin (Sigma, 1:50,000), NQO1 (Abcam), PGC-1α (Novus Biologicals), VDAC (Abcam), TFAM (generous gift from the Wallace Lab, 1:2,000), PRX3 (R&D systems 1:300), HMOX1 (OWL ID 58476). Secondary antibodies were used at 1:2,000, and blots were developed with Milipore Crescendo Western HRP Substrate.