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Protocol to quantify palmitoylation of cysteines in budding yeast
Yuqing Lei1, Jiangli Zhu2, Huihui Li1
1Department of Pathology, West China Second University Hospital, State Key Laboratory of Biotherapy, Sichuan University, Chengdu 610041, China.
STAR Protocols
|January 3, 2022
Summary
This study presents the acyl-biotin exchange (ABE) assay for detecting protein palmitoylation in yeast. This method uses biotinylated compounds to identify palmitoylated proteins for western blot analysis.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Palmitoylation is a critical post-translational modification.
- It involves the attachment of fatty acids to proteins, primarily targeting them to cellular membranes.
- Understanding palmitoylation is key to deciphering protein localization and function.
Purpose of the Study:
- To introduce a reliable protocol for assessing protein palmitoylation.
- To specifically detect cysteine-based palmitoylation in yeast.
- To enable downstream analysis of palmitoylated proteins.
Main Methods:
- The acyl-biotin exchange (ABE) assay is detailed.
- This method involves exchanging palmitoylation with biotinylated reagents.
- Palmitoylated proteins are affinity-purified and detected via western blot.
Main Results:
- The ABE assay provides a robust method for identifying palmitoylated cysteines.
- The protocol is demonstrated in yeast, offering a model system.
- Successful affinity purification and western blot detection are achieved.
Conclusions:
- The acyl-biotin exchange (ABE) assay is an effective tool for studying cysteine palmitoylation.
- This protocol is adaptable for use with other biological samples.
- It facilitates the investigation of protein lipidation and membrane targeting.
