Mohammed Jarvier N, Gelles Jesse D, Chipuk Jerry Edward
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.
Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.
STAR Protoc. 2022 Mar 18;3(2):101252. doi: 10.1016/j.xpro.2022.101252. eCollection 2022 Jun 17.
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).
BAX激活技术对于研究细胞凋亡的内在途径至关重要——数千种促凋亡信号汇聚于BAX激活过程。目前的方法主要局限于膜通透性研究，该研究评估寡聚化BAX的终点功能，但忽略了胞质BAX的早期激活步骤。在此，我们详细介绍FLAMBE：一种用于监测溶液中BAX早期激活的荧光偏振配体测定法。我们还描述了一种双指标参数化策略，用于在研究候选配体时提炼动力学数据并进行比较分析。有关本方案使用和执行的完整详细信息，请参阅Gelles等人（2022年）的文献。
