| 货号 | 8821S |
| 反应种属 | Human/Mouse |
| 应用 | W/IP |
| 目标/特异性 | Active Ras Detection Kit detects endogenous levels of GTP-bound (active) Ras 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). |
| 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. Boguski, M.S. and McCormick, F. (1993) Nature 366, 643-54. Avruch, J. et al. (1994) Trends Biochem Sci 19, 279-83. Buday, L. and Downward, J. (1993) Cell 73, 611-20. Huang, D.C. et al. (1993) Mol Cell Biol 13, 2420-31. Bos, J.L. (1989) Cancer Res 49, 4682-9. Ma, J. and Karplus, M. (1997) J Mol Biol 274, 114-31. |
Figure 1. NIH/3T3 cell lysates (500 µl at 1 mg/ml) were treated in vitro with GTPγS or GDP to activate or inactivate Ras (refer to optional step C in protocol). The lysates were then incubated with glutathione resin and GST-Raf1-RBD (lanes 2 and 3). GTPγS-treated lysate was also incubated without GST-Raf1-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 Ras mouse mAb. Anti-mouse IgG, HRP-linked Antibody #7076 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-PAK1-PBD (lanes 2 和3)孵育。GTPγS处理的裂解液也在缺乏GST-PAK1-PBD而存在谷胱甘肽树脂作为阴性对照的情况下孵育（lane4）。使用Ras Mouse mAb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-mouse IgG，HRP-linked Antibody #7076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。 |
