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.
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. 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 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.
Ras GTPase Chemi ELISA Kit FAQs
Can I use frozen cell or tissue samples as starting material?
For best results, we recommend using fresh material. This is an activity assay and freeze-thawing the starting material may mask experimental effects. If frozen sample is the only option, we recommend thawing and performing the homogenization step in the Complete Lysis Buffer. Whole cell extracts may be aliquoted and stored at -80°C. Avoid freeze/thaw cycles.
How do I prepare my sample if I am performing the assay with tissue?
For tissue, a modified protocol is required where a single-cell suspension is first created before the extraction. We recommend using the Nuclear Extract Kit, which contains a protocol for the preparation of whole-cell extract from tissue.
What types of Ras proteins will the included Ras antibody detect?
The H-Ras antibody will both detect H-Ras and K-Ras in human samples and H-Ras in mouse samples.
What controls are included in the kit?
The HeLa whole-cell extract (EGF treated) is provided as a positive control for Ras activation. Sufficient extract is supplied for 8 reactions per plate. This extract is optimized to give a strong signal when used at 25 µg/well.
Are there other positive or negative controls that can be used with the kit?
An optional protocol is included in the manual for using either GTPγS or GDP to create positive and negative controls from any whole cell extract. GTPγS acts as an activator while GDP acts as an inhibitor to Ras activation.
Ras GTPase Chemi ELISA Kit Publications
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Ras GTPase Chemi ELISA Kit Documents
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Ras GTPase Chemi ELISA Kit
1 x 96 rxns
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
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.
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.