Detection of Protein Palmitoylation Via Click Chemistry and Flow Cytometry

Palmitoylation is the process of attaching palmitate groups to protein cysteine residues. Many proteins have dynamic palmitoylation modifications that are added and removed to regulate protein function. Techniques to study protein palmitoylation, however, are slow and difficult to perform. We are in the process of developing a rapid technique for measuring protein palmitoylation using the palmitate analogue17‐ODYA and click chemistry followed by flow cytometry. To work out assay conditions, the Rho family GTPase TC10 is being used as a positive palmitoylation control, as it is known to be palmitoylated on its C‐terminal tail. To perform the analysis, 17‐ODYA is added to cells transfected with YFP‐tagged versions of TC10, and lysates from the cells are treated with click chemistry reagents to selectively conjugate the terminal alkyne group of 17‐ODYA with Alexa‐647 azide. The protein is isolated with magnetic anti‐FLAG beads which then are analyzed for YFP and Alexa‐647 fluorescence using flow cytometry. The current results indicate the method may be effectively detecting palmitoylation of TC10; however, the results will need to be validated using Western blotting as well as TC10 mutants that are incapable of being palmitoylated. Overall, the method will significantly enhance the time required to detect protein palmitoylation compared to traditional blot‐based techniques. Support or Funding Information This work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .