Measuring NLR oligomerization I: Size exclusion chromatography, co-immunoprecipitation, and cross-linking

Sonal Khare, Alexander D. Radian, Andrea Dorfleutner, Christian Stehlik*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

6 Scopus citations

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 crosslinking 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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages131-143
Number of pages13
DOIs
StatePublished - 2016

Publication series

NameMethods in Molecular Biology
Volume1417
ISSN (Print)1064-3745

Keywords

  • Caspase-1 activity assay
  • Co-immunoprecipitation
  • Cross-linking
  • Inflammasome
  • NLR
  • Nod-like receptor
  • Oligomerization
  • Protein-protein interaction
  • Size exclusion chromatography

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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