Immunoprecipitation of Flag-tagged CaV β2 Protein

Immunoprecipitation was performed using a Protein G Immunoprecipitation Kit (Sigma-Aldrich)^{6 (link)}. Cell pellet was resuspended in 1.0 mL of lysis buffer (20 mM sodium phosphate, 150 mM sodium chloride, 10% glycerol, 1 mM ethylenediaminetetraacetic acid, 0.5% Triton-X 100 [pH 7.2]) and complete TM protease inhibitor cocktail (Roche, Basel, Switzerland) at ∼ 1 mg/mL. The suspension was placed on ice for 1 h and centrifuged at 10,000×g at 4 °C for 15 min. The cleared lysate was incubated at 4 °C for 1 h with 2 μg of a monoclonal anti-flag antibody (Sigma-Aldrich) against flag-tagged Ca_Vβ2 protein. Protein G Sepharose (50 μL) was added to the samples, which were incubated for 16 h at 4 °C. The immunoprecipitate was washed five times with 1 mL of immunoprecipitation buffer before being eluted with 60 μL of Laemmli buffer. The eluted product (15 μL) and an equal volume of whole-cell lysate were subjected to 10% SDS-PAGE and Western blotting (ProBlot II AP; Promega) with an anti-β2 (Sigma-Aldrich), anti-GFP (GeneTex Inc., Irvine, CA), or anti-Ca_V1.2 (Alomone, Jerusalem, Israel) polyclonal antibody, which recognizes the intracellular loop between domains II and III of Ca_V1.2. Anti-rabbit IgG alkaline phosphatase conjugate (Promega) was used for immunodetection.