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

TL;DR

This paper describes a new method to detect palmitoylation in large synaptic proteins from rat neurons using an improved acyl-PEG labeling technique.

Contribution

An optimized APEGS assay is introduced for monitoring palmitoylation in high-molecular-weight proteins from primary neuronal cultures.

Findings

APEGS assay successfully detects palmitoylation in high-MW synaptic proteins.

Hydroxylamine cleavage and mPEG-MAL-10k labeling effectively label exposed cysteine residues.

Protocol includes culturing neurons and expressing tagged proteins for palmitoylation screening.

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

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).

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