fast, sensitive analysis of activated Ras GTPase
Ras GTPase Chemi ELISA Kit Overview
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:
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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.
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.
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.