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This volume provides methods and protocols on dynamin superfamily GTPases. Chapters are divided into three parts; detailing expression, purification, initial biochemical steps, undescribed analytical techniques, and biophysical and cellular characterization of DSPs in membrane remodeling, fission and fusion. 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.
Authoritative and cutting-edge, Dynamin Superfamily GTPases: Methods and Protocols to ensure successful results in the further study of this vital field.
List of contents
Isolation and Analysis of Mitochondrial Fission Enzyme DNM1 from Saccharomyces cerevisiae.- Purification of the Dynamin-related Protein Vps1 using Mammalian and Bacterial Expression Systems.- A Single Common Protocol for the Expression and Purification of Soluble Mammalian DSPs from Escherichia coli.- Affinity Purification and Functional Characterization of Dynamin-related Protein 1.- Purification and Characterization of MxB.- Purification of Farnesylated hGBP1 and Characterization of Its Polymerization and Membrane Binding.- Microscale Thermophoresis (MST) as a Tool for Measuring Dynamin Superfamily Protein (DSP)-lipid Interactions.- Nucleotide-dependent Dimerization and Conformational Switching of Atlastin.- Analysis of Mitochondrial Membrane Fusion GTPase OPA1 Expressed by the Silkworm Expression System.- Cell-free Analysis of Mitochondrial Fusion by Fluorescence Microscopy.- Electrophysiological MethodsFor Detection Of Membrane Leakage and Hemifission by Dynamin 1.- Integrating Optical and Electrochemical Approaches to Assess the Actions of Dynamin at the Fusion Pore.- Cellular Assays for Measuring Dynamin Activity in Muscle Cells.- Measuring Drp1 Activity in Mitochondrial Fission in vivo.- Quantifying Drp1-mediated Mitochondrial Fission by Immunostaining in Fixed Cells.- Imaging Dynamin-related Protein 1 (Drp1)-mediated Mitochondrial Fission in Living Cells.
