Functional assessment and quantitative kinetic analysis of BAX activation using large unilamellar vesicles

A myriad of diverse developmental and pro-death signals converge on the mitochondrial pathway of apoptosis, which is governed by the BCL‑2 family of proteins. Comprised of both pro- and anti-apoptotic family members, the BCL‑2 family functions to regulate mitochondrial outer membrane permeabilization (MOMP), often considered the "point of no return" in which a cell commits to an apoptotic outcome. Specifically, the effector BCL‑2 family proteins, BAX and BAK, are responsible for inducing MOMP and therefore investigations into their structural, cellular, and pharmacological regulation are critical to understanding the cellular commitment to apoptosis. A gold standard methodology for studying activation of BAX or BAK is the permeabilization of large unilamellar vesicles (LUVs), which are biochemically-defined model liposomes that mimic the major lipid composition of the outer mitochondrial membrane (OMM). Here, we provide a detailed protocol for generating LUVs containing a fluorescent dye/quencher pair to monitor real-time BAX activation and membrane permeabilization using a standard plate reader. Additionally, we detail example assay strategies to model interactions within the BCL‑2 family and provide a robust mathematical model for fitting and parameterizing kinetic LUV permeabilization data.