SNAREs - Methods and Protocols

This second edition details new and updated methods on soluble N-ethylmaleimide-sensitive factor attachment protein receptors( SNAREs) and their function are examined in the laboratory. Chapters guide readers through an overview of the basic properties of SNAREs, distribution and interaction with regulators of membrane fusion, activation of SNAREs in the priming stage by NSF/Sec18 and a-SNAP/Sec17, examining the structures and interactions of SNAREs, measuring the interactions of SNAREs, interactions of SNAREs, and post-translational modifications of SNAREs and how they affect function. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.



Authoritative and cutting-edge, SNAREs: Methods and Protocols, Second Edition aims to provide detailed methods so that novice as well as experienced researchers can explore the mechanisms of SNARE-mediated membrane fusion.

Inhaltsverzeichnis:

Molecular Dynamics Simulations of the SNARE Complex Interacting with Synaptotagmin, Complexin, and Lipid Bilayers.- Investigating complexin-membrane interactions using NMR and optical methods.- Molecular dynamics simulation for membrane fusion.- Use of Steered Molecular Dynamics to Explore the Conformational Changes of SNARE Proteins.- A Deep Learning and PSSM Profile Approach for Accurate SNARE Protein Prediction.- Use of Biotin-labeled Geranyl Pyrophosphate for Analysis of Ykt6 Geranylgeranylation.- Use of Bio-Layer Interferometry (BLI) to measure binding affinities of SNAREs and phosphoinositides.- Spectroscopic methods for detecting conformational changes during Sec18-Lipid interactions.- Assays of platelet SNARE-actin interactions.- Dissecting SNARE-Mediated Exocytosis in RBL-2H3 Mast Cells.- In vitro homotypic ER membrane fusion assay using isolated yeast microsomes.- Fluorescence anisotropy for monitoring cis- and trans-membrane interactions of synaptotagmin-1.- Structu ral Dynamics of SNARE Complex Assembly in the Ribbon Synapses Observed by smFRET.- FRAP assay to trace lipid mixing of the inner and outer leaflet of yeast vacuoles: Assessing the fusion state in live cells.- Mechanistic Insights into Synaptotagmin-1 Mediated Membrane Fusion and Interactions.- Negative staining electron microscopy of a highly flexible Sec1/Munc18 protein complex stabilized by glutaraldehyde crosslinking.- Electron tomography of organelles and vesicles in the investigation of SNARE function and localization.- A Dual-Color Fluorescence-based Ratiometric Assay to Measure Endocytic Trafficking of Surface Proteins.- SNARE-mediated membrane fusion probed using a synthetic organelle in the living bacterium.- Bimolecular Fluorescence Complementation (BiFC) Technique for exocytic proteins in murine hippocampal neurons.