Nonradioactive Analysis of Dynamic Protein Palmitoylation

Abstract

Methods to study protein S-palmitoylation dynamics have previously relied on metabolic labeling with [¹⁴C]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. Curr. Protoc. Protein Sci. 73:14.15.1-14.15.9. © 2013 by John Wiley & Sons, Inc.