Protein Arginylation

This volume presents a comprehensive overview of all the existing methods on analysing protein arginylation, from 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. This book also includes essays from the founders of the field, who originally discovered arginylation in the early 1960s and brought it to international recognition. 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, Protein Arginylation: Methods and Protocols would interest the emerging body of scientists involved in the studies of posttranslational arginylation and the rapidly growing community of researchers working on a broad range of posttranslational modifications, the analysis of which often meets similar challenges and utilizes similar principles as posttranslational arginylation.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts

Inhalt

Protein Arginylation: Over 50 Years 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 150k xg 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 -gal Degradation.- Bacterial Expression and Purification of Recombinant Arginyltransferase (ATE1) and Arg-tRNA Synthetase (RRS) for Arginylation Assays.- Assaying ATE1 Activity In Vitro.- High Throughput Arginylation Assay in Micro plate Format.- 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.- Applying Arginylation for Bottom-Up Proteomics.- Development of New Tools for the Studies of Protein Arginylation.