Titlebook: NLR Proteins; Methods and Protocol Francesco Di Virgilio,Pablo Pelegrín Book 2016 Springer Science+Business Media New York 2016 molecular i

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Assessing Extracellular ATP as Danger Signal In Vivo: The pmeLuc System,ogenous or endogenous danger signals by immune cells. Extracellular ATP is a ubiquitous danger signal released during septic or sterile inflammation. The development of reliable techniques to measure extracellular ATP in vivo has become an urgent need in inflammation studies after the discovery that

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

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Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immuns-associated speck-like protein containing a CARD (ASC). Following inflammasome activation, ASC polymerizes to form a large singular structure termed the ASC “speck,” which is crucial for recruitment of caspase-1 and its inflammatory activity. Hence, due to the considerably large size of these struc

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Measuring NLR Oligomerization III: Detection of NLRP3 Complex by Bioluminescence Resonance Energy T(. luciferase) and a fluorescent protein acceptor. BRET signal is dependent on the distance and the orientation between the donor and the acceptor and could be used to study protein–protein interactions and conformational changes within proteins in real time in living cells. This protocol describes

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Measuring NLR Oligomerization V: In Situ Proximity Ligation Assay,lex impedes detection of NLRP3 inflammasome formation by conventional biochemical and cell biology methods. In situ proximity ligation assay (PLA) provides an alternative method of detection, localization, and quantification of protein–protein interactions in tissue and cell samples. Two primary ant

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Assessing Caspase-1 Activation,s, the interleukin-1beta (IL-1β) and IL-18. In this chapter, we describe the technique of Western blot to assess caspase-1 activation. This method provides multiple information within one experiment. It allows the detection of both unprocessed and processed caspase-1 and substrates.

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Cell-Free Assay for Inflammasome Activation, cell-free system, based on cytosols of normally growing cells, the disruption of the cell membrane spontaneously activates the inflammasome. Studying the activation of the inflammasome in cytosolic extracts provides multiple advantages, as it is synchronized, rapid, strong, and mostly plasma membra

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