Active Rac1 Detection Kit

• 产品编号：CST-8815S      品牌：CST       原厂货号：8815S
• 产品分类：生化和细胞分析 > 细胞分析试剂盒 > 细胞结构分析试剂盒
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描述：

Active Rac1 Detection试剂盒可以识别内源性GTP-bound（active）Rac1如图1.通过内部测试，该试剂盒能够检测来自指定物种的蛋白，但也可能会检测到其他物种中的同源蛋白。.Active Rac1 Detection试剂盒提供了所有足以检测细胞中激活状态的Rac1 GTPase的试剂。GST-PAK1-PBD融合蛋白用以与激活状态的GTP-bound Rac1结合，随后通过谷胱甘肽树脂被免疫沉淀。Rac1的激活水平随后通过Rac1 Mouse mAb进行western blot分析以检测。小GTP-结合蛋白（G蛋白）Ras超家族包括了一大类蛋白（超过150个成员），基于其序列和功能相似性，可以被分为至少5组：Ras, Rho, Rab, Arf, 和 Ran (1-3).这些小G蛋白拥有GDP/GTP-结合和GTPase功能，能够在多种细胞和发育过程中发挥双向调控作用，包括细胞周期演进，细胞生存，细胞骨架重构，细胞极化和运动，囊泡和细胞核转运（1）。上游信号刺激GDP从GDP-结合形式（失活）解离，随后导致GTP结合形成GTP-结合形式（激活）。激活的G蛋白的下游效应结合区域经过构象变化，导致下游效应分子的结合和调控。这个激活状态能被GTPase关闭，它会水解GTP为GDP释放下游效应分子。这些Ras超家族蛋白固有的鸟苷酸交换和GTP水解活性也被鸟苷酸交换因子（GEFs）调节，GEFs会促进激活的GTP-结合形式和GTPase激活蛋白（GAPs）的形成，而GAPs会使GTPase变为GDP-结合失活形式（4）。Rac和Cdc42是Rho-GTPase家族的成员。在哺乳动物中，Rac有3种亚型，Rac1，Rac2，Rac3，它们的序列高度相似。Rac1和Cdc42，作为该家族中研究的最多的蛋白，表达非常广泛。Rac2表达在造血来源的细胞中，Rac3在大脑中高表达，也在其他多种组织中有表达。Rac和Cdc42蛋白在细胞骨架重构，细胞膜转运，转录调控，细胞生长和发育过程中都发挥了重要作用（5）。结合GTP后会激活Rac/Cdc42活性，GTP通过蛋白的GTPase功能水解为GDP，最终导致失活。GTP水解由GTPase激活蛋白（GAPs）协助，而GDP更换为GTP则通过鸟苷酸交换因子（GEFs）进行。RhoGDI，一个鸟苷酸解离抑制因子，会在另一个水平通过调节RhoGDI的结合来进行，鸟苷酸解离抑制因子会保留Rho家族GTPases处于失活的GDP结合状态，包括Rac和Cdc42（6,7）。

1. Takai, Y. et al. (2001) Physiol Rev 81, 153-208.
2. Colicelli, J. (2004) Sci STKE 2004, RE13.
3. Wennerberg, K. et al. (2005) J Cell Sci 118, 843-6.
4. Vigil, D. et al. (2010) Nat Rev Cancer 10, 842-57.
5. Wennerberg, K. and Der, C.J. (2004) J Cell Sci 117, 1301-12.
6. Bernards, A. and Settleman, J. (2004) Trends Cell Biol 14, 377-85.
7. Rossman, K.L. et al. (2005) Nat Rev Mol Cell Biol 6, 167-80.

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Specificity / Sensitivity

Active Rac1 Detection Kit detects endogenous levels of GTP-bound (active) Rac1 as shown in Figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Description

The Active Rac1 Detection Kit provides all reagents necessary for measuring activation of Rac1 GTPase in the cell. GST-PAK1-PBD fusion protein is used to bind the activated form of GTP-bound Rac1, which can then be immunoprecipitated with glutathione resin. Rac1 activation levels are then determined by western blot using a Rac1 Mouse mAb.

Background

The Ras superfamily of small GTP-binding proteins (G proteins) comprise a large class of proteins (over 150 members) that can be classified into at least five families based on their sequence and functional similarities: Ras, Rho, Rab, Arf, and Ran (1-3). These small G proteins have both GDP/GTP-binding and GTPase activities and function as binary switches in diverse cellular and developmental events that include cell cycle progression, cell survival, actin cytoskeletal organization, cell polarity and movement, and vesicular and nuclear transport (1). An upstream signal stimulates the dissociation of GDP from the GDP-bound form (inactive), which leads to the binding of GTP and formation of the GTP-bound form (active). The activated G protein then goes through a conformational change in its downstream effector-binding region, leading to the binding and regulation of downstream effectors. This activation can be switched off by the intrinsic GTPase activity, which hydrolyzes GTP to GDP and releases the downstream effectors. These intrinsic guanine nucleotide exchange and GTP hydrolysis activities of Ras superfamily proteins are also regulated by guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound form and GTPase activating proteins (GAPs) that return the GTPase to its GDP-bound inactive form (4).Rac and Cdc42 are members of the Rho-GTPase family. In mammals, Rac exists as three isoforms, Rac1, Rac2, and Rac3, which are highly similar in sequence. Rac1 and Cdc42, the most widely studied of this group, are ubiquitously expressed. Rac2 is expressed in cells of hematopoietic origin, and Rac3, while highly expressed in brain, is also found in many other tissues. Rac and Cdc42 play key signaling roles in cytoskeletal reorganization, membrane trafficking, transcriptional regulation, cell growth, and development (5). GTP binding stimulates the activity of Rac/Cdc42, and the hydrolysis of GTP to GDP through the protein's intrinsic GTPase activity, rendering it inactive. GTP hydrolysis is aided by GTPase activating proteins (GAPs), while exchange of GDP for GTP is facilitated by guanine nucleotide exchange factors (GEFs). Another level of regulation is achieved through the binding of RhoGDI, a guanine nucleotide dissociation inhibitor, which retains Rho family GTPases, including Rac and Cdc42, in their inactive GDP-bound state (6,7).

1. Takai, Y. et al. (2001) Physiol Rev 81, 153-208.
2. Colicelli, J. (2004) Sci STKE 2004, RE13.
3. Wennerberg, K. et al. (2005) J Cell Sci 118, 843-6.
4. Vigil, D. et al. (2010) Nat Rev Cancer 10, 842-57.
5. Wennerberg, K. and Der, C.J. (2004) J Cell Sci 117, 1301-12.
6. Bernards, A. and Settleman, J. (2004) Trends Cell Biol 14, 377-85.
7. Rossman, K.L. et al. (2005) Nat Rev Mol Cell Biol 6, 167-80.