E hexadec 2 en 15 ynal

Recombinant BAX (10 mM) was incubated with liposomes containing 0%, 5%, or 10% (E)-hexadec-2-en-15-ynal (Cayman Chemical) in the presence of BIM SAHB_A2 (10 μM) or vehicle (1% DMSO) for 2 h at room temperature. A click-chemistry reaction was then performed by adding 0.8 mM of premixed 1:1 CuSO₄:Tris((1-benzyl-4-triazolyl)methyl)amine (TBTA) complex, followed by 125 mM Azide-PEG3-biotin conjugate (Aldrich) and 1 mM sodium ascorbate. The reaction was incubated for 1 h at room temperature with gentle mixing. High-capacity streptavidin agarose beads (30 μL) were washed three times in 1 mL of liposomal release assay buffer containing 1% CHAPS. The beads were blocked in 3% BSA in PBS for 1 h at 4C, and then washed 3 times. The click reaction (1 μM) was added to the beads and rotated for 1 h at 4C. The beads were sequentially washed 3 times with liposomal buffer containing 1% CHAPS and then 3 times with PBS. Bound protein was eluted from the beads by adding 10 mg/mL biotin in 10% SDS and heating to 95°C for 10 min. Samples were prepared by adding 3x LDS loading dye containing 1M DTT to the elution mixture, followed by electrophoresis on a 4%–12% Bis-Tris gel and BAX western analysis using the HRP-BAX 2D2 antibody (Santa Cruz).