Protocol for detecting palmitoylation of high-molecular-weight rat synaptic proteins via acyl-PEG labeling
Year: 2024
Type: article
Abstract: 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 • Preparing primary rat cortical cell cultures • Expressing tagged proteins with lentiviral vectors • Detecting palmitoylation of high-MW synaptic proteins using a modified acyl-PEG assay • Screening palmitoylated proteins via size separation on SDS-PAGE Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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).
Source: STAR Protocols
Institution: University of Bristol
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,500
OpenAlex ID: W4402297750