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Methods to Probe Conformational Activation and Mitochondrial Activity of Proapoptotic BAK

  • Geetika Singh
  • Tudor Moldoveanu
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1877)

Abstract

Mitochondrial outer membrane permeabilization (MOMP) is a crucial initiating event in apoptosis that activates the caspase cascade to execute cell demise. The effector B-cell lymphoma 2 (BCL-2) antagonist killer (BAK) forms mitochondrial apoptotic pores to mediate MOMP. In healthy cells, BAK resides at the outer mitochondrial membrane as a dormant monomer. Upon direct interactions with the BCL-2 homology 3 (BH3)-only proapoptotic proteins during apoptosis, BAK undergoes conformational changes to form the active species associated with apoptotic pores. We describe methods to purify mitochondria for MOMP assays and to detect conformational changes in native BAK associated with MOMP by using limited proteolysis and cross-linking analyses.

Key words

Mitochondrial purification Membrane permeabilization Cytochrome c release Immunoblotting Conformational changes Limited proteolysis Cross-linking 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Geetika Singh
    • 1
    • 2
  • Tudor Moldoveanu
    • 1
    • 2
  1. 1.Department of Structural BiologySt. Jude Children’s Research HospitalMemphisUSA
  2. 2.Department of Chemical Biology and TherapeuticsSt. Jude Children’s Research HospitalMemphisUSA

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