A kinetic fluorescence polarization ligand assay for monitoring BAX early activation

FLAMBE: A kinetic fluorescence polarization assay to study activation of monomeric BAX

“…The metric, Polarization ( P ), is calculated from the parallel (∥) and perpendicular (⊥) intensities ( I ) for each time point ( t ). Data are reproduced and adapted from Gelles et al. (2022) .…”

“…Investigators will have to consult the manufacturer's documentation to set up an analogous protocol to measure fluorescence polarization and adjust the steps within this protocol for their plate reader. Investigators may purchase commercially available recombinant human BAX protein, but the preferred method is to express and purify recombinant BAX protein in-house ( Suzuki et al., 2000 ; Gelles et al., 2022 ). Unlabeled BAK-BH3 peptide (AnaSpec Cat#: AS-61616) may be purchased and conjugated to a different fluorescent label at the investigator's discretion provided the plate reader has a compatible FP cube installed or available.…”

“…Investigators may purchase commercially available recombinant human BAX protein, but the preferred method is to express and purify recombinant BAX protein in-house ( Suzuki et al., 2000 ; Gelles et al., 2022 ).…”

“…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) .…”

“…For complete details on the use and execution of this protocol, please refer to Gelles et al. (2022) .…”