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

Jarvier N Mohammed; Jesse D Gelles; Jerry Edward Chipuk

doi:10.1016/j.xpro.2022.101252

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

STAR Protoc. 2022 Mar 18;3(2):101252. doi: 10.1016/j.xpro.2022.101252. eCollection 2022 Jun 17.

Authors

Jarvier N Mohammed^{1

2

3

4

5}, Jesse D Gelles^{1

2

3

4}, Jerry Edward Chipuk^{1

2

3

4

6

5}

Affiliations

¹ Laboratory 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.
² Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
³ Department of Dermatology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
⁴ The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
⁵ The Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
⁶ The Diabetes, Obesity, and Metabolism Institute, 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).

Keywords: Cell Biology; Cell-based Assays; Molecular Biology; Molecular/Chemical Probes; Signal Transduction; Single-molecule Assays.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Apoptosis* / physiology
Cytosol / metabolism
Fluorescence Polarization
Kinetics
bcl-2-Associated X Protein / metabolism

Substances

bcl-2-Associated X Protein

Abstract

Publication types

MeSH terms

Substances

Grants and funding