Format

Send to

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

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

Author information

1
Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, McGaw Pavilion Suite M-300, 240 E Huron, Chicago, IL, 60611, USA.
2
Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, McGaw Pavilion Suite M-300, 240 E Huron, Chicago, IL, 60611, USA. c-stehlik@northwestern.edu.

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

PMID:
27221486
PMCID:
PMC5058782
DOI:
10.1007/978-1-4939-3566-6_8
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Springer Icon for PubMed Central
Loading ...
Support Center