BCL-2 Family Proteins: Methods and Protocols

Description

This volume explores numerous techniques for the genetic, molecular, biochemical, and structural examination of BCL-2 family proteins and their interactions. The chapters in this book cover topics such as the relevance of BCL-2 proteins in health and disease; evaluating cellular dependencies to specific BCL-2 family proteins; flow-cytometry-based methods for measuring BCL-2 proteins and mitochondrial-based cell death; measuring activity and interactions of BCL-2 family proteins in the presence of mitochondria, artificial membranes or yeast; conformational activation and oligomerization of pro-apoptotic proteins BAX and BAK leading to cytochrome c release and apoptosis; structural and biophysical studies in solution and lipid vesicles using nuclear magnetic resonance, cryo-electron microscopy, fluorescence microscopy and electron paramagnetic resonance. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, BCL-2 Family Proteins: Methods and Protocols is a valuable resource to inspire and encourage novice and established scientists to further their research and make new discoveries in this exciting field. Front Matter ....Pages i-xi Overview of BCL-2 Family Proteins and Therapeutic Potentials (Jason D. Huska, Heather M. Lamb, J. Marie Hardwick)....Pages 1-21 Database and Bioinformatic Analysis of BCL-2 Family Proteins and BH3-Only Proteins (Abdel Aouacheria, Vincent Navratil, Christophe Combet)....Pages 23-43 Application of Mito-Priming to Generate BCL-2 Addicted Cells (Jonathan Lopez, Stephen W. G. Tait)....Pages 45-60 BH3 Profiling: A Functional Assay to Measure Apoptotic Priming and Dependencies (Cameron Fraser, Jeremy Ryan, Kristopher Sarosiek)....Pages 61-76 Flow Cytometry-Based Detection and Analysis of BCL-2 Family Proteins and Mitochondrial Outer Membrane Permeabilization (MOMP) (Lindsey M. Ludwig, Katrina L. Maxcy, James L. LaBelle)....Pages 77-91 Investigating BCL-2 Family Protein Interactions in Yeast (Stéphen Manon)....Pages 93-109 Liposomal Permeabilization Assay to Study the Functional Interactions of the BCL-2 Family (Denis E. Reyna, Evripidis Gavathiotis)....Pages 111-119 Mitochondrial Isolation and Real-Time Monitoring of MOMP (Mark P. A. Luna-Vargas, Jarvier N. Mohammed, Jesse D. Gelles, Jerry Edward Chipuk)....Pages 121-130 Photocrosslinking Approach to Investigate Protein Interactions in the BCL-2 Family (Jialing Lin, Arthur E. Johnson, Zhi Zhang)....Pages 131-149 Assessment of Dynamic BCL-2 Protein Shuttling Between Outer Mitochondrial Membrane and Cytosol (Joachim Lauterwasser, Franziska Fimm-Todt, Frank Edlich)....Pages 151-161 Quantification of BCL-2 Family Members by Flow Cytometry (Morey L. Smith, Stephen K. Tahir)....Pages 163-172 Methods to Probe Calcium Regulation by BCL-2 Family Members (Marcos A. Carpio, Samuel G. Katz)....Pages 173-183 Methods to Probe Conformational Activation and Mitochondrial Activity of Proapoptotic BAK (Geetika Singh, Tudor Moldoveanu)....Pages 185-200 Probing BAK and BAX Activation and Pore Assembly with Cytochrome c Release, Limited Proteolysis, and Oxidant-Induced Linkage (Sweta Iyer, Rachel T. Uren, Ruth M. Kluck)....Pages 201-216 BCL-2 Protein Family Interaction Analysis by Nuclear Magnetic Resonance Spectroscopy (Thomas P. Garner, Evripidis Gavathiotis)....Pages 217-231 Reconstitution and Characterization of BCL-2 Family Proteins in Lipid Bilayer Nanodiscs (Yong Yao, Francesca M. Marassi)....Pages 233-246 Cryo-Electron Microscopy to Study Bax Pores and MOMP (Tomomi Kuwana)....Pages 247-256 CW EPR and DEER Methods to Determine BCL-2 Family Protein Structure and Interactions: Application of Site-Directed Spin Labeling to BAK Apoptotic Pores (Tirtha Mandal, Eric J. Hustedt, Likai Song, Kyoung Joon Oh)....Pages 257-303 Rapid Imaging of BCL-2 Family Interactions in Live Cells Using FLIM-FRET (Elizabeth J. Osterlund, Nehad Hirmiz, Christian Tardif, David W. Andrews)....Pages 305-335 Quantification of the Interactions Between BCL-2 Proteins by Fluorescence Correlation Spectroscopy (Fabronia Murad, Ana J. Garcia-Saez)....Pages 337-350 Isolation of Synthetic Antibodies Against BCL-2-Associated X Protein (BAX) (Zhou Dai, Jonathan R. Lai)....Pages 351-357 Back Matter ....Pages 359-360