Protocol to identify S-acylated proteins in hippocampal neurons using ω-alkynyl fatty acid analogs and click chemistry

Abstract

Summary: S-acylation, commonly palmitoylation, is the addition of fatty acids to cysteines to regulate protein localization and function. S-acylation detection has been hampered by limited sensitivity and selectivity in low-protein, costly samples like cultured neurons. Here, we present a protocol for sensitive and selective bioorthogonal labeling and click-chemistry-based detection of S-acylated proteins in primary hippocampal neurons. We describe steps for metabolically labeling neurons with alkynyl fatty acid, click chemistry, NeutrAvidin-based capture, and elution with hydroxylamine. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell culture
• Cell separation/fractionation
• Molecular/Chemical Probes