Monoclonal anti v5 antibody

Lysates containing equal amounts of protein as determined by the Bradford assay or bicinchoninic acid assay (BCA assay) were combined with EZview Red anti-FLAG M2 affinity gel (Sigma, F2426) as per the manufacturer’s instructions. For V5-AMPKγ1 immunoprecipitation, protein A/G agarose (Santa Cruz, sc-2003) was combined with monoclonal anti-V5 antibody (Sigma, V8012), which was used at 1ug of antibody/1 mg of protein, and rotated overnight at 4°C. The supernatant was removed and the affinity gel or agarose pellets were washed by adding 1mL of 0.1% NP40 lysis buffer followed by 5 minutes rotation at 4°C for a total of 4 times. For immunoprecipitation using the membrane fraction, affinity gel pellets were washed with 1mL of 0.1% NP40 lysis buffer, followed by 5 minutes rotation at 4°C for 3 times and with 1mL 0.5% NP40 lysis buffer followed by 5 minutes rotation at 4°C for one time. Detergent was then removed from the membrane fraction using Pierce Detergent spin columns. FLAG-K1 and/or FLAG-K1 domain deleted mutants were eluted using 3X FLAG peptide (Sigma, F4799) as per the manufacturer’s instructions. Laemmli buffer (2X) was added to the FLAG-K1eluate or directly to the V5-AMPKγ1/agarose samples (1:1) and all samples were heated at 100°C for 6 minutes. Proteins were resolved by SDS-PAGE followed by Western blot.

Equal amounts of cells grown at 37°C in LB medium were harvested during exponential phase. Cells were pelleted, resuspended in loading buffer, and separated on a NuPAGE Bis-Tris gel (Thermo Fisher). After transfer, membranes were blotted with monoclonal anti-V5 antibody (Sigma-Aldrich) or anti-RpoB antibody (BioLegend). RpoB was blotted as a loading control. Pierce ECL Western blotting substrate (Thermo Scientific) was added before exposing the X-ray film. Experiments were carried out in at least biological triplicates.