Blue Native Polyacrylamide Gel Electrophoresis to Monitor Inflammasome Assembly and Composition
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 wordsInflammasome Blue Native PAGE 2D Native/SDS-PAGE NAIP NLRC4 Flagellin
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.
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