TMBIM6 Immunoprecipitation Protocol

TMBIM6-HA was immunoprecipitated by the soft elution method^{61 (link)}. Briefly, cell lysates were prepared in lysis buffer composed of 20 mM Tris-HCl (pH 8.0), 135 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 1% Triton X-100, and complete protease and phosphatase inhibitor cocktails (Sigma-Aldrich). Crude lysates (500 μg for each immunoprecipitation) were incubated with antibody (1–2 μg) for 6 h at 4 °C, followed by addition of protein A/G sepharose beads (Sigma-Aldrich) and incubation for an additional 1 h. Immunoprecipitates were washed five times with phosphate-buffered saline (PBS) containing 0.1% Triton X-100 or PBS before SDS–PAGE and immunoblotting.