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

Bushra P Yucel; Jeremy M Henley; Kevin A Wilkinson

doi:10.1016/j.xpro.2024.103296

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

Bushra P Yucel^*, Jeremy M Henley^*, Kevin A Wilkinson^*

^*Corresponding author for this work

Research output: Contribution to journal › Protocol

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.

Original language	English
Article number	103296
Number of pages	16
Journal	STAR Protocols
Volume	5
Issue number	3
Early online date	6 Sept 2024
DOIs	https://doi.org/10.1016/j.xpro.2024.103296
Publication status	Published - 20 Sept 2024

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Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.

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Roles of protein SUMOylation in AMPA receptor trafficking, synaptic dysfunction and cognitive impairment in dementia
Henley, J. M. (Principal Investigator)
1/03/14 → 30/06/18
Project: Research

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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.",

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AB - 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.

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

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Roles of protein SUMOylation in AMPA receptor trafficking, synaptic dysfunction and cognitive impairment in dementia

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