Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases.
Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases.
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DOI：
10.1007/978-1-4939-8612-5_13
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Buranda T
中科院分区：
文献类型：
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作者：
Simons P;Bondu V;Wandinger-Ness A;Buranda T
小型单体三磷酸鸟嘌呤水解酶 (GTPase) 是普遍存在的细胞信号传导整合剂。信号激活 GTP 酶，然后 GTP 酶与效应分子结合，在细胞内传递信号。 GTPase 效应陷阱流式细胞术 (G-Trap) 利用基于微珠的蛋白质固定和双色流式细胞术快速定量测量细胞或组织裂解物中的 GTPase 活性状态。从使用红色（700 nm）波长的分级荧光强度进行颜色编码的商业细胞复合物珠组开始，珠组被衍生化，通过此处描述的详细方案在表面上显示谷胱甘肽。然后可以将不同的谷胱甘肽-S-转移酶效应蛋白（GST 效应蛋白）附着到每组的表面。对于该测定，用户可以单独孵育珠子组或以多重形式与裂解物一起孵育，以便从单个样品中快速、选择性地捕获活性的、GTP 结合的 GTP 酶。之后，使用流式细胞术根据红色珠子强度鉴定珠子携带的 GTP 酶，并使用与绿色荧光团缀合的单克隆抗体或通过标记的二抗检测每个珠子的活性 GTP 酶量。提供了三个例子来说明效应器功能化珠子在测量来自少于 50,000 个细胞的单个裂解物中至少五种 GTP 酶的激活的功效。
Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700 nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-S-transferase-effector protein (GST-effector protein) can then be attached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borne GTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells.