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.