Advertisement

Blue Native Polyacrylamide Gel Electrophoresis to Monitor Inflammasome Assembly and Composition

  • Eric M. Kofoed
  • Russell E. Vance
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1040)

Abstract

Inflammasomes are large cytosolic multi-protein complexes that form in response to infection and other stimuli, and play an important role in the innate immune response. Traditional methods for assaying inflammasome activation have relied on detection of autoproteolysis of the cysteine protease caspase-1, or proteolytic processing of its substrate, the inflammatory cytokine IL-1β. These measurements report on the final output of inflammasome activation, but do not assess inflammasome assembly and composition. Here we describe the application of blue native gel electrophoresis as a tool for investigating stimulus-dependent inflammasome assembly and for identification of individual components within the multi-protein complex.

Key words

Inflammasome Blue Native PAGE 2D Native/SDS-PAGE NAIP NLRC4 Flagellin 

Notes

Acknowledgments

E.K. would like to thank Dr. Maryanne McClellan who taught Western blotting in an era of in-house ECL solution when X-ray film was developed by hand, and Dr. Elizabeth Vierling and Dr. Gary Lee who taught E.K. native PAGE a decade ago. Genentech kindly provided the α-NLRC4 antibody, and Bill Dietrich contributed the α-NAIP5 antibody.

References

  1. 1.
    Bonardi V, Cherkis K, Nishimura MT, Dangl JL (2012) A new eye on NLR proteins: focused on clarity or diffused by complexity? Curr Opin Immunol 24(1):41–50. doi: S0952-7915(11)00181-6 [pii]  10.1016/j.coi.2011.12.006 PubMedCrossRefGoogle Scholar
  2. 2.
    Bergsbaken T, Fink SL, Cookson BT (2009) Pyroptosis: host cell death and inflammation. Nat Rev Microbiol 7(2):99–109. doi: nrmicro2070 [pii]  10.1038/nrmicro2070 PubMedCrossRefGoogle Scholar
  3. 3.
    Martinon F, Burns K, Tschopp J (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell 10(2):417–426. doi: S1097276502005993 [pii] PubMedCrossRefGoogle Scholar
  4. 4.
    Schagger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199(2):223–231PubMedCrossRefGoogle Scholar
  5. 5.
    Wittig I, Braun HP, Schagger H (2006) Blue native PAGE. Nat Protoc 1(1):418–428. doi: nprot.2006.62 [pii]  10.1038/nprot.2006.62 PubMedCrossRefGoogle Scholar
  6. 6.
    Lasserre JP, Menard A (2012) Two-dimensional Blue Native/SDS gel electrophoresis of multiprotein complexes. Methods Mol Biol 869:317–337. doi: 10.1007/978-1-61779-821-4_27 PubMedCrossRefGoogle Scholar
  7. 7.
    Schagger H, Cramer WA, von Jagow G (1994) Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal Biochem 217(2):220–230. doi: S0003269784711122 [pii] PubMedCrossRefGoogle Scholar
  8. 8.
    Kofoed EM, Vance RE (2011) Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity. Nature 477(7366):592–595. doi: [pii] 10.1038/nature10394 PubMedCrossRefGoogle Scholar
  9. 9.
    Susnea I, Bernevic B, Wicke M, Ma L, Liu S, Schellander K, Przybylski M (2012) Application of MALDI-TOF-mass spectrometry to proteome analysis using stain-free gel electrophoresis. Top Curr Chem. doi: 10.1007/128_2012_321 Google Scholar
  10. 10.
    Swamy M, Kulathu Y, Ernst S, Reth M, Schamel WW (2006) Two dimensional Blue Native-/SDS-PAGE analysis of SLP family adaptor protein complexes. Immunol Lett 104(1–2):131–137. doi: S0165-2478(05)00350-0 [pii]  10.1016/j.imlet.2005.11.004 PubMedCrossRefGoogle Scholar
  11. 11.
    Swamy M, Siegers GM, Minguet S, Wollscheid B, Schamel WW (2006) Blue native polyacrylamide gel electrophoresis (BN-PAGE) for the identification and analysis of multiprotein complexes. Sci STKE 2006(345):pl4. doi: stke.3452006pl4 [pii]  10.1126/stke.3452006pl4 PubMedGoogle Scholar

Copyright information

© Springer Science+business media, New York 2013

Authors and Affiliations

  • Eric M. Kofoed
    • 1
  • Russell E. Vance
    • 1
  1. 1.Division of Immunology and Pathogenesis, Department of Molecular and Cell BiologyUniversity of CaliforniaBerkeleyUSA

Personalised recommendations