Measuring NLR Oligomerization I: Size Exclusion Chromatography, Co-immunoprecipitation, and Cross-Linking

Methods Mol Biol. 2016;1417:131-43. doi: 10.1007/978-1-4939-3566-6_8.

Abstract

Oligomerization of nod-like receptors (NLRs) can be detected by several biochemical techniques dependent on the stringency of protein-protein interactions. 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 fast protein liquid chromatography (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. Chemical cross-linking covalently joins two or more molecules, thus capturing the oligomeric state with high sensitivity and stability. Apoptosis-associated speck-like protein containing a caspase activation domain (ASC) oligomerization has been successfully used as readout for NLR or AIM2-like receptor (ALR) inflammasome activation in response to various pathogen- or damage-associated molecular patterns (PAMPs or 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: Caspase-1 activity assay; Co-immunoprecipitation; Cross-linking; Inflammasome; NLR; Nod-like receptor; Oligomerization; Protein–protein interaction; Size exclusion chromatography.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / chemistry*
  • Animals
  • CARD Signaling Adaptor Proteins / chemistry*
  • Chromatography, Gel
  • Chromatography, Liquid
  • Cross-Linking Reagents
  • Humans
  • Immunoprecipitation
  • Macrophages / cytology
  • Mice
  • NLR Family, Pyrin Domain-Containing 3 Protein / chemistry*
  • Protein Multimerization
  • Staphylococcal Infections / metabolism*
  • Staphylococcus aureus / physiology
  • THP-1 Cells

Substances

  • Adaptor Proteins, Signal Transducing
  • CARD Signaling Adaptor Proteins
  • Cross-Linking Reagents
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • NLRP7 protein, human
  • PYCARD protein, human