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Full Description
This book provides detailed protocols and untold tips and tricks regarding the most well-known examples of proximity labeling methods, in which the protein of interest is genetically fused to or labeled with an enzyme that can generate short-lived reactive species to non-specifically label molecules within a certain radius of up to twenty nanometers. Beginning with peroxidase-based proximity labeling methods, the volume continues with BioID, proximity labeling methods that describe the proximity ligation assay to detect RNA-DNA interactions, UV cross-linking to demonstrate RNA-protein interactions, and how chemical and enzymatic reactivities can be improved upon DNA-DNA and protein-protein interactions, as well as "proximity-induced self-labeling," where the radius of labeling is zero. Written for the highly successful Methods in Molecular Biology series, 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 practical, Proximity Labeling: Methods and Protocols serves as an ideal guide for researchers exploring the crucial roles that proximity-driven reactions play in biological systems.
Contents
The EMARS Reaction for Proximity Labeling.- Proteomic Proximity Labelling to Reveal Interactions Between Biomolecules.- Establishing Cell Culture-based Experimental Setups for Proximity Labeling Using Ascorbate Peroxidase (APEX).- APEX Peroxidase Catalyzed Proximity Labeling and Multiplexed Quantitative Proteomics.- Identification of Lipid Droplet Proteomes by Proximity Labeling Proteomics Using APEX2.- Antibody-driven Proximity Labeling in Fixed Tissues.- BioID: A Method to Generate a History of Protein Associations.- Direct Identification of Biotinylated Proteins from Proximity Labeling (Spot-BioID).- Identifying Synaptic Proteins by in vivo BioID from Mouse Brain.- A Proximity Ligation-based Method to Detect RNA-DNA Association.- Enzymatic Transfer of Photo-cross-linkers for RNA-Protein Photo-cross-linking at the mRNA 5'-cap.- Proximity-induced Bioorthogonal Chemistry Using Inverse Electron Demand Diels-Alder Reaction.- Overcoming the Limitations of Sortase with Proximity-based Sortase-mediated Ligation (PBSL).- Proximity-driven Site-specific and Covalent Labeling of Proteins with a TexasRed Fluorophore Reacting (ReacTR) Peptide Tag.- Affinity Conjugation for Rapid and Covalent Labeling of Proteins in Live Cells.- Ligand-directed N-Sulfonyl Pyridone Chemistry for Selective Native Protein Labeling and Imaging in Live Cell.
