Monoclonal anti his6 antibody

Bacterial strains were grown either in liquid cultures or on swarm agar plates in M63 minimal salts medium supplemented with 1 mM MgSO₄, 0.2% glucose, and 0.5% casamino acids (CAA) with 0.5% arabinose for induction of plasmid-based expression of the pMotC::His₆ protein. Whole cell lysates and membrane fractions were prepared as previously described (58 (link)). Total protein concentrations in membrane fractions were quantified using the Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). For Western blotting, equivalent total protein quantities from membrane samples were resolved by SDS-PAGE using Any kD polyacrylamide gels (Bio-Rad, Hercules, CA). Proteins transferred to a nitrocellulose membrane were probed with a monoclonal anti-His₆ antibody (Qiagen, Germantown, MD). Detection of proteins via Western blotting was performed by fluorescence detection using IR-Dye-labeled fluorescent secondary antibodies and imaged using the Odyssey CLx Imager (LICOR Biosciences, Inc., Lincoln, NE). Protein quantification was performed using Image Studio Lite software (LICOR Biosciences, Inc., Lincoln, NE).

The recombinant CB₂ protein was detected by Western blot with mouse monoclonal antibody against human cannabinoid receptor CB₂ (R&D Systems) or monoclonal anti-His6 antibody (Qiagen). The Western blot was developed with anti-mouse HRP antibody (1:5000 dilution) and visualized by chemiluminescence with Gel Logic imaging system (Kodak). The concentration of the detergent-solubilized protein was determined with a UV–Vis spectrometer (Agilent Technologies) using DC Protein Assay Reagent (BioRad), and bovine serum albumin (BSA) as protein standard.