Methods to Measure NLR Oligomerization I: Size Exclusion Chromatography, Co-immunoprecipitation, and Cross-Linking

Abstract

Protein oligomerization is a common principle of regulating cellular responses. Oligomerization of NLRs is essential for the formation of NLR signaling platforms and can be detected by several biochemical techniques. Some of these biochemical methods can be combined with functional assays, such as caspase-1 activity assay. Size exclusion chromatography (SEC) allows separation of native protein lysates into different sized complexes by FPLC for follow-up analysis. Using co-immunoprecipitation (co-IP), combined with SEC or on its own, enables subsequent antibody-based purification of NLR complexes and associated proteins, which can then be analyzed by immunoblot and/or subjected to functional caspase-1 activity assay. Native gel electrophoresis also allows detection of the NLR oligomerization state by immunoblot. Chemical cross-linking covalently joins two or more molecules, thus capturing the oligomeric state with high sensitivity and stability. ASC oligomerization has been successfully used as readout for NLR/ALR inflammasome activation in response to various PAMPs and DAMPs in human and mouse macrophages and THP-1 cells. Here, we provide a detailed description of the methods used for NLRP7 oligomerization in response to infection with Staphylococcus aureus (S. aureus) in primary human macrophages, co-immunoprecipitation, and immunoblot analysis of NLRP7 and NLRP3 inflammasome complexes as well as caspase-1 activity assays. Also, ASC oligomerization is shown in response to dsDNA, LPS/ATP, and LPS/nigericin in mouse bone marrow-derived macrophages (BMDMs) and/or THP-1 cells or human primary macrophages.

Keywords: Blue native electrophoresis; Caspase-1 activity assay; Co-immunoprecipitation; Cross-linking; Inflammasome; NLR; Nod-like receptor; Oligomerization; Protein-protein interaction; Size exclusion chromatography.