| 货号 | 8820S |
| 描述 | The Active Rho Detection Kit provides all reagents necessary for measuring activation of Rho GTPase in the cell. GST-Rhotekin-RBD fusion protein is used to bind the activated form of GTP-bound Rho, which can then be immunoprecipitated with glutathione resin. Rho activation levels are then determined by western blot using a Rho Rabbit Antibody.Active Rho Detection试剂盒提供了所有足以检测细胞中激活状态Rho GTPase的试剂。GST-Rhotekin-RBD融合蛋白用于与GTP-bound Rho的激活状态结合，随后可以被谷胱甘肽树脂免疫沉淀。Rho的激活水平随后使用一个Rho Rabbit 抗体通过western blot实验检测。 |
| 反应种属 | Human/Mouse |
| 应用 | W/IP |
| 目标/特异性 | Active Rho Detection Kit detects endogenous levels of GTP-bound (active) Rho 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. |
| 供应商 | CST |
| 背景 | 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). 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）。 |
| 存放说明 | -80C |
| 参考文献 | Takai, Y. et al. (2001) Physiol Rev 81, 153-208. Colicelli, J. (2004) Sci STKE 2004, RE13. Wennerberg, K. et al. (2005) J Cell Sci 118, 843-6. Vigil, D. et al. (2010) Nat Rev Cancer 10, 842-57. DerMardirossian, C. and Bokoch, G.M. (2005) Trends Cell Biol 15, 356-63. Wennerberg, K. and Der, C.J. (2004) J Cell Sci 117, 1301-12. Wheeler, A.P. and Ridley, A.J. (2004) Exp Cell Res 301, 43-9. |
Figure 1. NIH/3T3 cell lysates (500 µl at 1 mg/ml) were treated in vitro with GTPγS or GDP to activate or inactivate Rho (refer to optional step C in protocol). The lysates were then incubated with glutathione resin and GST-Rhotekin-RBD (lanes 2 and 3). GTPγS-treated lysate was also incubated without GST-Rhotekin-RBD in the presence of glutathione resin as a negative control (lane 4). Western blot analysis of cell lysate (20 µg, lane 1) or 20 µl of the eluted samples (lanes 2, 3, and 4) was performed using a Rho Rabbit Antibody. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.图1. NIH/3T3细胞提取物（500 µl at 1 mg/ml）在体外使用GTPγS或 GDP处理以激活或失活Rac1（参考步骤C）。这些裂解液随后被谷胱甘肽树脂和GST-Rhotekin-RBD (lanes 2 和3)孵育。GTPγS处理的裂解液也在缺乏GST-Rhotekin-RBD而存在谷胱甘肽树脂作为阴性对照的情况下孵育（lane4）。使用Rho Rabbit 抗体对细胞裂解液(20 µg, lane 1)或20ul 稀释样品（lanes 2, 3, 和 4)）进行western blot分析。Anti-rabbit IgG, HRP-linked Antibody #7074作为二抗。 | |
Figure 2. The GTP-bound GTPase pull-down process can be divided into 3 steps as shown. Step 1: Mix sample, binding protein, and glutathione resin in the spin cup and incubate at 4ºC to allow GTP-bound GTPase binding to the glutathione resin through GST-linked binding protein. Step 2: Remove unbound proteins by centrifugation. Step 3: Elute glutathione resin-bound GTPase with SDS buffer. The eluted sample can then be analyzed by western blot.图2.如图所示， GTP-bound GTPase pull-down过程可以被分成3个步骤。第一步：在自旋杯中混合样品，结合蛋白，和谷胱甘肽树脂，4ºC孵育以使GTP-bound GTPase与谷胱甘肽树脂通过GST- linked结合蛋白结合。第二步：通过离心去除没有结合的蛋白。第三步：使用SDS缓冲液稀释谷胱甘肽树脂。稀释的样品可以随后用于western blot分析。 |
