Figure 7.

Workflow for crosslinking and reconstitution of refolded VDAC1 into membrane vesicles for functional assays

(A) Lipid stocks in chloroform are dried down in a round bottom flask, resuspended in aqueous buffer, and extruded to produce unilamellar, monodisperse liposomes.

(B) Purified VDAC1 is buffer-exchanged to remove Tris and crosslinked using EGS, an amino-reactive covalent crosslinker.

(C) Liposomes and crosslinked (or mock-treated) protein samples are treated with detergent and incubated together for 1 h before the overnight addition of Bio-Beads. Resultant proteoliposomes reconstituted with crosslinked or mock-treated VDAC1, and liposomes containing no protein, can be tested for channel and scramblase activity via fluorescence-based assays.