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

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

STAR protocols (2024-05-19)
Charlotte A Townsend, Andrey A Petropavlovskiy, Jordan A Kogut, Alysha M Church, Shaun S Sanders
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
APT1 Inhibitor, palmostatin B, The APT1 Inhibitor, palmostatin B controls the biological activity of APT1.
Sigma-Aldrich
Anti-Pan-Ras Antibody, clone RAS 10, clone RAS 10, from mouse