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Full Description
This second edition provides updated and new chapters that detail comprehensive overview of all the existing methods on analyzing protein arginylation. Chapters guide readers through the early methods utilizing crude protein preparations and whole-cell assays to the latest advanced methods involving recombinant protein techniques, antibodies, high precision mass spectrometry, and chemical probes. 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 methods, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.
Cutting-edge and thorough, Protein Arginylation: Methods and Protocols, Second Edition aims to be of interest to novice researchers involved in posttranslational arginylation research and researchers working on a broad range of posttranslational modifications.
Contents
Protein arginylation: milestones of discovery.- Recollection of how we came across the protein modification with amino acids by aminoacyl tRNA-protein transferase.- Arginyltransferase: a personal and historical perspective.- Arginylation in a Partially Purified Fraction of 150,000xg Supernatants of Axoplasm and Injured Vertebrate Nerves.- Preparation of ATE1 enzyme from native mammalian tissues.- Correlated measurement of endogenous ATE1 activity on native acceptor proteins in tissues and cultured cells to detect cellular aging.- Assaying the Post-translational Arginylation of Proteins in Cultured Cells.- Assaying ATE1 activity in yeast by β-gal degradation.- Assaying arginylation activity in cell lysates using a fluorescent reporter.- Assaying intracellular arginylation activity using a fluorescent reporter.- Bacterial expression and purification of recombinant arginyltransferase (ATE1) and Arg-tRNA synthetase (RRS) for arginylation assays.- Preparation of tRNAArg for arginylation assay by in vitro transcription.- Preparation of an enriched tRNAArg fraction for arginylation by expression in E. coli.- Enzymatic aminoacylation of tRNAArg using recombinant Arg-tRNA synthetase.- Assaying ATE1 activity in vitro.- High throughput arginylation assay in microplate format.- Assaying for arginyltransferase activity and specificity by peptide arrays.- Assay of arginyltransferase activity by a fluorescent HPLC method.- Identification of arginylated proteins by mass spectrometry.- Analysis of arginylated peptides by subtractive Edman degradation.- Transferase-Mediated Labeling of Protein N-Termini with Click Chemistry Handles.- Synthesis of peptides and proteins with site-specific glutamate arginylation.- Reconstitution of the arginyltransferase (ATE1) iron-sulfur cluster.- N-terminal arginylation pulldown analysis using the R-catcher tool.- Identification of protein arginylation by encapsulated N-terminal peptide enrichmentmethod.- Monitoring the Activation of Selective Autophagy via N-terminal Arginylation.- Analyzing the Interaction of Arginylated Proteins and Nt-Arg-Mimicking Chemical Compounds to N-Recognins.- Synthesis of Stably Charged Arg-tRNAArg for Structural Analysis.- A High-Throughput Colorimetric Microplate Assay for Determination of Plasma Arginase Activity.- Development of new tools for the studies of protein arginylation.
