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  3. Click-chemistry-based protocol for detecting 4-octyl-itaconate-alkylated proteins in primary mouse macrophages

Click-chemistry-based protocol for detecting 4-octyl-itaconate-alkylated proteins in primary mouse macrophages

  • STAR Protoc. 2024 Sep 18;5(4):103314. doi: 10.1016/j.xpro.2024.103314.
Chaofei Su 1,  Tian Cheng 2,  Hanyi Zhang 2,  Hang Yin 3
Affiliations

Affiliations

  • 1 State 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. Electronic address: scf19@mails.tsinghua.edu.cn.
  • 2 State 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.
  • 3 State 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. Electronic address: yin_hang@tsinghua.edu.cn.
PMID: 39298323 DOI: 10.1016/j.xpro.2024.103314
Abstract

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.

Keywords

Cell-based Assays; Chemistry; Immunology; Molecular/Chemical Probes.

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