Utilizing FLAMBE to study the structure-function relationship and regulation of monomeric BAX activation in solution.

The BCL‑2 family member BAX is responsible for inducing mitochondrial outer membrane permeabilization (MOMP) and committing a cell to apoptosis. Specifically, native BAX is an inactive cytosolic monomer, which, upon activation by direct activator BCL‑2 family proteins, undergoes intramolecular rearrangements and structural changes, resulting in BAX translocation to the mitochondrial outer membrane, oligomerization, and MOMP - collectively, we refer to this progression of structural conformers as the BAX activation continuum. In vitro BAX activation studies are invaluable tools for investigating BAX structure-function biology and studying cellular and pharmacological modulators of apoptosis; however, such methodologies are commonly limited to endpoint functional phenotypes (e.g., membrane permeabilization) and thus overlook molecular regulation of soluble monomeric BAX. To address this methodological gap, we developed FLAMBE (a fluorescence polarization ligand assay for monitoring BAX early-activation), a solution-based kinetic assay that infers BAX activation via the concomitant dissociation of a labeled BH3 peptide. FLAMBE maintains the benefits of rapid kinetic data generation in an economical microplate format using commercially available reagents without requiring specialized equipment or large quantities of protein. Herein, this protocol describes how to perform FLAMBE assays, the recommended optimization workflow, and a dual-metric parameterization strategy to distill kinetic data for comparative analyses.