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- 英文名：
Ras GTPase Chemi ELISA Kit
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Active Motif
- 规格：
1 x 96 rxns
Ras GTPase Chemi ELISA Kit Overview
The Ras GTPase Chemi ELISA Kit was designed specifically for the study of Ras activation, and can be used to detect and quantify activated Ras GTPase in cell or tissue extracts. Monitoring levels of activated Ras can be useful for studying novel Ras activating signaling pathways, detecting oncogenic Ras proteins, or monitoring the effects of possible therapeutic compounds.
The Ras GTPase Chemi ELISA Kit uses the unique binding properties of the Ras-binding domain of the c-Raf kinase protein (Raf-RBD) which only binds activated Ras. A GST-fused Raf-RBD protein is bound to a 96-well glutathione-coated assay plate and addition of sample to the plate results in the binding of activated Ras to the Raf-RBD. A primary antibody specific for Ras is then added to the individual wells, followed by an HRP-conjugated secondary antibody and developing reagent . The plate is then read on a luminometer, which provides a sensitive chemiluminescent readout of activated Ras. The 96-well plate is suitable for both manually assaying small numbers of samples and for use in automated high-throughput screening assays.
The Ras GTPase Chemi ELISA Kit uses the unique binding properties of the Ras-binding domain of the c-Raf kinase protein (Raf-RBD) which only binds activated Ras. A GST-fused Raf-RBD protein is bound to a 96-well glutathione-coated assay plate and addition of sample to the plate results in the binding of activated Ras to the Raf-RBD. A primary antibody specific for Ras is then added to the individual wells, followed by an HRP-conjugated secondary antibody and developing reagent . The plate is then read on a luminometer, which provides a sensitive chemiluminescent readout of activated Ras. The 96-well plate is suitable for both manually assaying small numbers of samples and for use in automated high-throughput screening assays.
Flow Chart of Ras GTPase Chemi ELISA Kit(Click image to enlarge)
Ras GTPase Background
The Ras protein is a GTPase enzyme. GTPases are a family of enzymes which bind to and hydrolyze GTP, functioning as molecular switches. When bound to GTP, Ras is active and activates effector proteins involved in the downstream control of cell proliferation and differentiation. When bound to GDP, Ras is inactive.Ras signaling cascades are normally only transiently activated. This is because the Ras GTPase activity gradually converts GTP to GDP, rendering the protein inactive. However, oncogenic mutations of the Ras gene can lock the protein into the active GTP conformation, creating a form of the protein that is always active. The result is constituatively active Ras signaling and cells which grow uncontrollably. In fact, mutations of the Ras gene are present in around 30% of human cancers and are present in an especially high percentage of pancreatic, lung, and colorectal cancers. Consequently, Ras has become a popular target for cancer research and anti-cancer therapeutics.
Ras GTPase Chemi ELISA Kit Highlights
- Detection Range – Works with 3-25 µg whole cell extract or > 0.6 ng purified protein per well.
- Better Results – ELISA format is more sensitive than pull-down/Western methods.
- Versatile – Use with extracts from cell and tissue samples, or with purified Ras protein
- Reactivity – detects activated human K-Ras and H-Ras or activated rodent H-Ras
- Quick – Results in fewer than 5 hours
Ras GTPase Chemi ELISA Kit Contents
The Ras GTPase Chemi ELISA Kit contains sufficient reagents to perform 96 reactions. It is shipped on dry ice and contains reagents with multiple storage temperatures inside. Please confirm receipt of all reagents upon arrival and store items at the appropriate temperatures as indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. This kit includes the following components:- Hela whole-cell extract (EGF treated); Store at -80°C
- GST-Raf-RBD; Store at -80°C
- H-Ras antibody; Store at -20°C
- Anti-rat HRP-conjugated IgG; Store at -20°C
- Protease Inhibitor Cocktail (PIC); Store at -20°C
- Lysis/Binding Buffer AM1; Store at 4°C
- 10X Wash Buffer AM2; Store at 4°C
- 10X Antibody Binding Buffer AM2; Store at 4°C
- Chemiluminescent Reagent; Store at 4°C
- Reaction Buffer; Store at 4°C
- 96-well Glutathione-coated Assay Plate; Store at 4°C
- Plate Sealer; Store at 4°C
Ras GTPase Chemi ELISA Data
Figure 1: Ras GTPase Chemi ELISA quantifies activated Ras
Increasing amounts of whole-cell extract from HeLa cells that had been stimulated with 5 ng/ml of EGF for 2 minutes were assayed using the Ras GTPase Chemi ELISA Kit. Data is also shown for unstimulated HeLa cells which contain basal levels of activated Ras.
Increasing amounts of whole-cell extract from HeLa cells that had been stimulated with 5 ng/ml of EGF for 2 minutes were assayed using the Ras GTPase Chemi ELISA Kit. Data is also shown for unstimulated HeLa cells which contain basal levels of activated Ras.
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文献和实验相关实验
Ras是大鼠肉瘤(rat sarcoma,Ras)的英文缩写。Ras蛋白是原癌基因 c—ras的表达产物，相对分子质量为21kDa，属单体 GTP结合蛋白，具有弱的 GTP酶活性。Ras蛋白的活性状态对细胞的生长、分化、细胞骨架、蛋白质运输和分泌等都具有影响，其活性则是通过与GTP或GDP的结合进行调节。 Ras的活性受两个蛋白的控制,一个是鸟苷交换因子(guanine nucleotide exchange factor, GEF),它的作用是促使GDP从Ras蛋白
仪（北京科美东雅生物技术有限公司生产）。 1.3 试剂 乙肝标志物酶法检测试剂由上海科华生物工程股份有限公司提供，批号20080810；化学发光法为北京科美东雅生物技术有限公司提供的配套试剂，批号20081208。 1.4 方法 对上述标本分别用ELISA法和化学发光法进行检测，均严格按照各自的试剂盒说明书操作。 1.5 数据处理 使用SPSS13.0统计软件对两种方法检测结果进行χ 2检验。 2 结果 两种方法对乙型肝炎病毒标志物的对比检测结果见表
Mathematical Investigation of How Oncogenic Ras Mutants Promote Ras Signaling
We have used a mathematical model of the Ras signaling network to link observable biochemical properties with cellular levels of RasGTP. Although there is abundant data characterizing Ras biochemistry, attributing specific changes
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Ras GTP酶化学发光ELISA试剂盒
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