2. Academic Validation
  3. Protocol for reconstitution of oligomeric assembly of NAIP5-NLRC4 inflammasome in vitro

Protocol for reconstitution of oligomeric assembly of NAIP5-NLRC4 inflammasome in vitro

  • STAR Protoc. 2023 Sep 20;4(4):102581. doi: 10.1016/j.xpro.2023.102581.
Xiurong Wu 1,  Jiahuai Han 2
Affiliations

Affiliations

  • 1 State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian 361102 China. Electronic address: [email protected].
  • 2 State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian 361102 China; Research Unit of Cellular Stress of CAMS, Xiang'an Hospital of Xiamen University, Cancer Research Center of Xiamen University, School of Medicine, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian 361102 China. Electronic address: [email protected].
PMID: 37733592 DOI: 10.1016/j.xpro.2023.102581
Abstract

Inflammasomes are multimeric protein complexes that have crucial functions in innate immunity. Here, we present a protocol to reconstitute the PELO-driven assembly of NAIP5-NLRC4 inflammasome in vitro. We describe steps for expression and purification of recombinant PELO and flagellin, preparation of native cell lysate containing NAIP5-NLRC4, and in vitro assembly of NAIP5-NLRC4 inflammasome. We then detail analysis of NAIP5-NLRC4 inflammasome by blue native polyacrylamide gel electrophoresis and immunoblotting. This protocol can be adapted to monitor the oligomeric assembly of other inflammasome types. For complete details on the use and execution of this protocol, please refer to Wu et al. (2023).1.

Keywords

Chemistry; Immunology; Protein Expression and Purification.

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