Protocol for the visualization and identification of S-palmitoylated proteins in HCT-116 cells using metabolic...

Reversible S-palmitoylation facilitates the regulation of a plethora of protein functions. Here, we present a protocol for enriching lipidated proteins with click-chemistry labeling and immunoprecipitation using biotin-azide-streptavidin interaction where protein activity assays are possible. We describe steps for performing the copper-based reaction for high-scale applications in milligram ranges, sample preparation, and the elution of palmitoylated-biotinylated proteins. Further, we detail procedures for the quantification of S-palmitoylation of isolated membrane proteins via reporter fluorophore on the western-blot level without lipid background. For complete details on the use and execution of this protocol, please refer to Merz et al.¹.