Measuring NLR Oligomerization I: Size Exclusion Chromatography, Co-immunoprecipitation, and Cross-Linking.
Measuring NLR Oligomerization I: Size Exclusion Chromatography, Co-immunoprecipitation, and Cross-Linking.
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DOI：
10.1007/978-1-4939-3566-6_8
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发表时间：
2016
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影响因子：
--
通讯作者：
Stehlik, Christian
中科院分区：
文献类型：
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作者：
Khare, Sonal;Radian, Alexander D;Dorfleutner, Andrea;Stehlik, Christian
NLR的寡聚化可以通过几种生物化学技术检测，这取决于蛋白质-蛋白质相互作用的严格性。这些生物化学方法中的一些可以与功能测定（例如胱天蛋白酶-1活性测定）组合。尺寸排阻色谱（SEC）允许通过FPLC将天然蛋白裂解物分离成不同大小的复合物用于后续分析。使用免疫共沉淀（co-IP），与SEC组合或单独使用，能够随后基于抗体纯化NLR复合物和相关蛋白，然后可以通过免疫印迹分析和/或进行功能性半胱天冬酶-1活性测定。化学交联共价连接两个或多个分子，从而以高灵敏度和稳定性捕获低聚状态。ASC寡聚化已成功地用作响应于人和小鼠巨噬细胞和THP-1细胞中的各种PAMP和DAMP的NLR/ALR炎性体活化的读出。在这里，我们提供了用于NLRP 7寡聚化的方法的详细描述，以响应感染金黄色葡萄球菌（金黄色葡萄球菌）在原代人巨噬细胞，共免疫沉淀和免疫印迹分析NLRP 7和NLRP 3炎性体复合物以及半胱天冬酶-1活性测定。此外，在小鼠骨髓衍生的巨噬细胞（BMDM）和/或THP-1细胞或人原代巨噬细胞中，显示ASC寡聚化响应于dsDNA、LPS/ATP和LPS/尼日利亚菌素。
Oligomerization of 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 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. ASC oligomerization has been successfully used as readout for NLR/ALR inflammasome activation in response to various PAMPs and 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 Stapylococcus 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.