title: Nonradioactive analysis of dynamic protein palmitoylation.
abstract: Methods to study protein S-palmitoylation dynamics have previously relied on metabolic labeling with [(14)C]palmitate, which requires additional safety precautions and long exposures. Nonradioactive alkynyl palmitate analogs have been developed for in-gel fluorescence detection and affinity purification. Cells metabolically labeled with the commercially available analog 17-octadynoic acid are lysed and then combined with azide-linked reporter tags for efficient conjugation by copper-catalyzed click chemistry in phosphate buffer. This approach has been demonstrated to label hundreds of endogenous palmitoylated proteins and is compatible with traditional pulse-chase methods. This protocol describes the reagents and procedures for labeling and detection of dynamic palmitoylation in mammalian cells.
authors: Department of Chemistry, University of Michigan, Ann Arbor, Michigan.
authors: Brent R
authors: BR
authors: Martin
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D057930:Click Chemistry; D054878:Lipoylation; D008322:Mammals; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
chemical_list: D011506:Proteins
keywords: click chemistry; palmitoylation; post-translational modifications