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Updated: Jun 12, 2025
Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry
Published on: April 9, 2021
Click-chemistry-based protocol for detecting 4-octyl-itaconate-alkylated proteins in primary mouse macrophages
Chaofei Su1, Tian Cheng1, Hanyi Zhang1
1State Key laboratory of Membrane Biology, School of Pharmaceutical Sciences, Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing 100084, China.
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4-Octyl itaconate (4-OI), a derivative of itaconate, inhibits inflammation by alkylating its target proteins. Here, we present a click-chemistry-based protocol for detecting 4-OI-alkylated proteins in mouse primary bone-marrow-derived macrophages (BMDMs) by using an itaconate-alkyne (ITalk) probe. We describe steps for culturing and treating BMDMs and details on using click chemistry in the cell lysate. We also detail procedures for detecting alkylated proteins by western blot. For complete details on the use and execution of this protocol, please refer to Su et al.1.
