Visualization of multiprotein complexes by flow cytometry.
Visualization of multiprotein complexes by flow cytometry.
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DOI：
10.1002/0471142735.im0509s87
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发表时间：
2009-11
影响因子：
--
通讯作者：
Schrum, Adam G
中科院分区：
文献类型：
--
作者：
Schrum, Adam G
多蛋白质复合体和其他蛋白质-蛋白质相互作用在几乎所有的细胞过程中都扮演着重要的角色。用流式细胞术分析蛋白质复合体的免疫共沉淀(IP-FCM，或“Fly-p”方法)提供了一种灵敏的手段来测量天然/非变性状态下的这些相互作用。首先，免疫沉淀抗体被共价偶联到聚苯乙烯乳胶珠上，其低自体荧光与流式细胞仪兼容。这些抗体偶联的珠子用于免疫沉淀细胞裂解物中存在的特定蛋白质(初级分析物)。最后，用针对相互作用伙伴或可能与主要分析物相关的二级分析物的氟铬偶联抗体来探测与珠子相关的蛋白质复合体。使用定量FACS方法可以将产生的半定量荧光数据转换为珠子上共结合分子的估计数量。该方法代表了一种稳健的技术，可以在不需要基因工程或大样本的情况下评估天然蛋白质-蛋白质相互作用。
Multiprotein complexes and other protein-protein interactions play important roles in virtually all cellular processes. Analysis of co-immunoprecipitation of protein complexes by flow cytometry (IP-FCM, or “the fly-p” method) provides a sensitive means to measure these interactions in the native/non-denatured state. First, immunoprecipitating antibodies are covalently coupled to polystyrene latex beads whose low autofluorescence is compatible with flow cytometry. These antibody-coupled beads are used to immunoprecipitate a specific protein (primary analyte) present in cell lysates. Finally, the protein complexes associated with the beads are probed with fluorochrome-conjugated antibodies specific for interaction partners, or secondary analytes, that may be associated with the primary analyte. The use of quantitative FACS methodology can allow the semi-quantitative fluorescence data generated to be converted into estimated numbers of co-associated molecules on the beads. The method represents a robust technique to assess native protein-protein interactions without requiring genetic engineering or large sample sizes.