Protocol for detecting palmitoylation of high-molecular-weight rat synaptic proteins via acyl-PEG labeling

Here, we present an optimized acyl-PEGyl exchange gel shift (APEGS) assay to monitor palmitoylation of high-molecular-weight proteins from primary neuronal cultures. We describe steps for culturing cortical neurons from rat embryos and expressing proteins of interest. We then detail procedures for employing a fatty acyl exchange technique wherein hydroxylamine is used to cleave palmitic acid from the palmitoyl-thioester bond, exposing cysteine residues that are subsequently labeled with methoxy polyethylene glycol maleimide (mPEG-MAL-10k). For complete details on the use and execution of this protocol, please refer to Yucel et al.¹.