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FLAMBE: A kinetic fluorescence polarization assay to study activation of monomeric BAX
Jarvier N Mohammed1,2,3,4,5, Jesse D Gelles1,2,3,4, Jerry Edward Chipuk1,2,3,4,6,5
1Laboratory of Mitochondrial Biology in Human Health and Disease, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
Abstract:
BAX activation techniques are crucial to studying the intrinsic pathway of apoptosis- thousands of pro-apoptotic signals converge on BAX activation. Current methodologies are predominantly limited to membrane permeabilization studies, which assess endpoint functionality of oligomeric BAX, but overlook early activation steps of cytosolic BAX. Here we detail FLAMBE: a fluorescence polarization ligand assay for monitoring BAX early-activation in solution. We also describe a dual-metric parameterization strategy for distillation of kinetic data and comparative analyses when studying candidate ligands. For complete details on the use and execution of this protocol, please refer to Gelles et al. (2022).
