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

Abstract

Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell-based Assays
• Immunology
• Molecular/Chemical Probes
• Chemistry