Protocol for the visualization and identification of S-palmitoylated proteins in HCT-116 cells using metabolic labeling via click chemistry

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Reversible S-palmitoylation is a critical post-translational modification regulating diverse protein functions.
• Understanding palmitoylation dynamics is essential for deciphering cellular signaling and protein activity.

Purpose of the Study:

• To present a robust protocol for enriching lipidated proteins.
• To enable subsequent protein activity assays and quantification of S-palmitoylation.

Main Methods:

• Utilizing click-chemistry labeling with biotin-azide and streptavidin-biotin interaction for enrichment.
• Performing copper-based reactions for high-scale applications (milligram range).
• Developing procedures for sample preparation, elution, and western blot quantification using reporter fluorophores.

Main Results:

• Successful enrichment of palmitoylated-biotinylated proteins.
• Demonstrated feasibility of protein activity assays post-enrichment.
• Established a method for quantifying S-palmitoylation on isolated membrane proteins without lipid background.

Conclusions:

• The presented protocol offers a versatile approach for studying S-palmitoylation.
• This method facilitates the investigation of palmitoylation's role in protein function and regulation.
• The protocol is suitable for high-scale applications and detailed analysis of membrane proteins.