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Ras GTPase Chemi ELISA Kit

fast, sensitive analysis of activated Ras GTPase

Active Motif's Ras GTPase Chemi ELISA Kit is the first ELISA-based kit designed to detect and quantify activated Ras GTPase. Because of its ELISA format, it is much more sensitive than pull-down/Western methods. This enables the detection of low levels of protein, while also using less sample. In addition, ELISAs are more quantitative than Westerns, and they eliminate the need to run and develop gels.

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: Flowchart of GTPase process.

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