Activation, Isolation, and Analysis of the Death-Inducing Signaling Complex.
Activation, Isolation, and Analysis of the Death-Inducing Signaling Complex.
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死亡诱导信号复合物的激活、分离和分析。
DOI：
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发表时间：
2015
影响因子：
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通讯作者：
M. MacFarlane
中科院分区：
文献类型：
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作者：
M. Hughes;C. Langlais;K. Cain;M. MacFarlane
该协议描述了使用亲和纯化对 CD95 (APO-1/Fas) 死亡诱导信号复合物 (DISC) 进行激活、分离和分析。使用生物素标记的抗 CD95 抗体实现激活，并使用链霉亲和素珠捕获天然 DISC 复合物。这种方法最大限度地减少了所涉及的步骤数量以及使用未标记抗体和 Protein A/G 珠进行免疫沉淀时常见的抗体的任何潜在非特异性相互作用或交叉反应性。通过蛋白质印迹分析分离的复合物的组成，以鉴定已知的 DISC 成分，并且可以通过检测 caspase-8 裂解产物来确认 DISC 中 procaspase-8 的二聚化诱导的自催化加工。 DISC 相关的 caspase-8 激活 caspase 级联的潜力可以通过测量荧光底物 Ac-IETD.AFC 的 caspase-8 依赖性裂解或通过使用外源蛋白底物进行生物测定来确定。
This protocol describes activation, isolation, and analysis of the CD95 (APO-1/Fas) death-inducing signaling complex (DISC) using affinity purification. Activation is achieved using a biotin-labeled anti-CD95 antibody and the native DISC complex is captured using streptavidin beads. This approach minimizes both the number of steps involved and any potential nonspecific interactions or cross-reactivity of antibodies commonly seen in immunoprecipitations using unlabeled antibodies and protein A/G beads. Composition of the isolated complex is analyzed via western blot to identify known DISC components, and dimerization-induced autocatalytic processing of procaspase-8 at the DISC can be confirmed by detection of caspase-8 cleavage products. The potential for DISC-associated caspase-8 to activate the caspase cascade can be determined by measuring caspase-8-dependent cleavage of the fluorigenic substrate Ac-IETD.AFC, or by performing a bioassay using exogenous protein substrates.