Ras GTPase活性检测ELISA试剂盒

快速、灵敏地检测激活态Ras GTPase

 

Ras GTPase Chemi ELISA kit 概览

Ras GTPase Chemi ELISA Kit
 

Ras GTPase Chemi ELISA试剂盒是专门为研究激活态Ras而设计的,可用于检测和定量细胞或组织提取物中激活态的Ras GTPase。监测激活的Ras水平有助于研究新的Ras激活信号通路,检测致癌Ras蛋白,或评估可能的治疗性化合物的效果。

Ras GTPase Chemi ELISA试剂盒利用c-Raf激酶蛋白(Raf RBD)Ras结合域的独特结合特性,该蛋白仅结合被激活的Ras。GST融合的Raf RBD蛋白结合到96孔谷胱甘肽涂层的分析板上,向板上添加样品使激活态的Ras与Raf RBD结合。然后将Ras特异性一级抗体添加到各个孔中,紧接着添加HRP结合的二级抗体和显影试剂。然后在光度计上读取该板,该光度计可提供激活Ras的灵敏化学发光读数。96孔板的设计既适用于手动分析少量样品,也适用于自动高通量筛选分析。

Flow Chart of Ras GTPase Chemi ELISA Kit

Flow Chart of Ras GTPase Chemi ELISA Kit
(Click image to enlarge)

Ras GTPase 背景介绍

Ras蛋白是一种GTPase酶。GTP酶是一种能够结合和水解GTP的酶家族,起着分子开关的作用。当与GTP结合时,Ras具有活性并激活参与细胞增殖和分化下游控制的效应蛋白。当与GDP结合时,Ras处于非活动状态。

Ras信号级联通常仅短暂激活。这是因为Ras GTPase活性随着GTP转化为GDP,蛋白质逐渐失去活性。然而,Ras基因的致癌突变可以将蛋白质锁定在活性GTP构象中,从而形成一种始终活跃的蛋白质形式。结果是组成性活跃的Ras信号和细胞生长不受控制。事实上,Ras基因突变存在于约30%的人类癌症中,尤其是胰腺癌、肺癌和结直肠癌中。因此,Ras已成为癌症研究和抗癌治疗的热门靶点。 Learn more about targeting mutant Ras proteins in our Blog: Undruggable No More - Advances for Targeting Mutant Ras Proteins and Cancer.

Ras GTPase Chemi ELISA试剂盒的亮点

  • 适用范围 – 每孔3-25 μg 全细胞提取物,或>0.6 ng 的纯化后蛋白
  • 更好的结果 – ELISA原理,比Pull-down/Western更加准确灵敏
  • 多用途 – 可使用细胞,组织提取物,以及纯化后的Ras蛋白
  • 反应性 – 可检测人源K-Ras,H-Ras或鼠源的H-Ras
  • 高效率 – 不到5小时即可获得结果
 

Ras GTPase Chemi ELISA试剂盒组分

Ras GTPase Chemi ELISA试剂盒包含足够的试剂,可完成96个反应。我们采用干冰运输,其中并包含具有多种存储温度的试剂。请在收货时确认收到所有试剂,并将物品储存在如下所示的适当温度下。所有试剂在按要求保存的情况下,可保持稳定长达6个月。该试剂盒包括以下组分:

  • 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 数据展示

Ras GTPase Chemi ELISA quantifies activated Ras

图1:Ras GTPase Chemi ElISA 检测激活态RAS
使用Ras GTPase Chemi ELISA kit 检测用5 ng/ml EGF刺激2分钟的HeLa细胞的全细胞提取物。数据还显示了含有本底水平的未受刺激的HeLa细胞的Ras活性强度。

 

Ras GTPase Chemi ELISA试剂盒常见问题

是否能够使用冻存的组织或细胞做为起始样本?

为了获得更加准确的结果,我们建议使用新鲜的组织或细胞样本。这是个活性检测实验,冻存或经过融冻的样本可能会屏蔽实验效果。若冻存样本是唯一选择,我们建议在Complete Lysis buffer中解冻并完成均质化步骤。全细胞提取物可等分并储存在-80°C。避免反复的冷冻/解冻。

若我使用组织样本,该如何开始我的实验呢?

针对组织样本,则需对实验步骤进行一定的修改,在蛋白提取之前,需将其制备成单细胞悬液。我们建议使用核提取试剂盒,该试剂盒包含从组织制备全细胞提取物的完整方案。

哪些类型的Ras蛋白可被Ras抗体检测到?

H-Ras抗体可检测到人员的H-Ras和K-Ras,以及鼠源的H-Ras。

试剂盒中的对照是什么呢?

本试剂盒提供HeLa全细胞提取物(EGF处理后)作为Ras激活的阳性对照。提供足够的萃取物,每个板可进行8次反应。该提取物经过优化,以在25µg/孔的条件下使用时提供强信号。

有其它的阳性对照或阴性对照可用于此试剂盒吗?

实验手册中包含了建议,使用GTPγS或GDP作为任何全细胞提取物中的阳性和阴性对照的可选方案。GTPγS是Ras激活的激活子,而GDP是Ras激活的抑制子。

 

Ras GTPase Chemi ELISA试剂盒引用文章

搜索我们的数据库,查看使用我们的Ras GTPase Chemi ELISA试剂盒的客户发表的文章。


 
 
 

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Name Format Cat No. Price  
Ras GTPase Chemi ELISA Kit 1 x 96 rxns 52097 ¥11,380 Buy

Ras GTPase Chemi ELISA advantages

  • More sensitive – assay uses only 25 µg of extract, which is 20-fold less than pull-down/Western methods
  • Better results – quantitative readouts make it easier to compare results
  • Less effort – no need to run gels or develop Western blots
  • Save time – results in less than 5 hours
  • Versatile – assay activated extracts from cells or tissue samples, or study recombinant Ras protein
 
Quantification of activated Ras in stimulated HeLa cells using the Ras GTPase Chemi ELISA Kit
 
Figure 1: Quantification of activated Ras in stimulated HeLa cells.

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.

Small GTPases, also known as GTP-binding proteins, are a family of enzymes that serve as molecular switches in regulating a number of signal transduction pathways including cellular growth, apoptosis and differentiation. These switches regulate by alternating between active (GTP-bound) and inactive (GDB-bound) states. Ras GTPase is of particular interest as aberrant regulation by Ras has been implicated in a number of disease states.

The Ras GTPase Chemi ELISA method

Because activated Ras binds specifically to the Ras-binding domain (RBD) of Raf effector protein, Raf-RBD can be used as a probe to isolate Ras-GTP. The Ras GTPase Chemi ELISA Kit contains a Raf-RBD protein that is fused to GST and a 96-well, glutathione-coated assay plate. GST-Raf-RBD is first incubated on the plate for one hour to immobilize the capture probe. 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 and quantitative chemiluminescent readout of activated Ras.

 
Flow chart of the Ras GTPase Chemi ELISA Kit process
 
Figure 1: Flow chart of the Ras GTPase Chemi ELISA Kit process.

A protein containing the binding domain for Ras GTPase is bound to the wells of a 96-well plate. Addition of sample results in binding of activated Ras, which is then quantified using a Ras antibody, HRP-conjugated secondary antibody and developing solution on a luminometer.

Contents & Storage

96-well glutathione-coated assay plate with plate sealer, GST-Raf-RBD, primary antibody, HRP-conjugated secondary antibody, positive control cell extract, Protease Inhibitor Cocktail, Lysis and Reaction buffers. Reagent storage conditions vary from room temperature to -80°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.