Description :
The small GTPases are molecular switches which control cellular processes such as cytoskeletal re-organization, axonal guidance, vesicle trafficking, budding in yeast, gene expression and cell motility. The balance of the GTP to GDP bound state underlies the switch mechanism as they turn from an activated (GTP form) to inactive state (GDP form). The balance of GTP to GDP bound states is controlled by catalytic proteins that either increase the rate of exchange of GDP for GTP (GEFs), increase the GTPase activity (GAPs), or prevent the exchange of GDP (GDIs). Recently it has been shown that the GAP family of proteins is large (70 members) and potentially important for changing a cell from a normal to disease status.
GAP activity can be reduced by deletion or mutation in cells; in this case the small GTPase targeted by the GAP has an extended time in the active GTP state, in essence creating a permanently active state. For example the ability of Rho GTPase to control axonal guidance is exemplified in the finding that mutations of a Rho-GAP can lead to mental retardation. In cancer, mutated Ras-GAPs (NF1 gene) have been shown to cause neurofibromatosis, and mutated Rheb-GAP has been shown to cause tuberous sclerosis complex.
Although some GAPs are known to have GAP activity, the majority of assumed GAP proteins have only been implicated by homology to contain GAP activity. Cytoskeleton Inc. is facilitating the exploration of this field by introducing the GAP Assay Kit. Several small GTPase proteins (Ras, RhoA, CDC42 and Rac1) are included such that the researcher can screen the small G-proteins for GAP like activity which is usually small G-protein specific i.e. RhoGAP or Ras GAP. It is likely that new domain information will identify other small G-protein GAPs which have not been apparent. In addition the new assay kits are easily adapted for High Throughput Screen format which allows development of ligands for pharmaceutical studies. Please inquire for bulk reagent quotes if you are considering a screening application.
The reagents in this kit have been optimized to enable high activity from your GAP protein such that you can detect the enhanced GTPase of a small G-protein through a simple absorbance based detection method. The small G-protein is incubated in the presence of GAP protein (Rho GAP is the control protein included in this kit), GTP and the optimized buffer. The overall GTPase activity of small G-proteins is composed of two components which limit the activity; these are a) endogenous GTPase activity and b) GDP dissociation rate. The endogenous activity can be enhanced by the addition of a suitable GAP protein, whereas the dissociation rate can be enhanced by buffer optimization.
Usage :
1. Determination of the activity of uncharacterized GAPs.
2. Biochemical characterization of small GTPases and their associated GAPs.
3. Examination of the regulation of GAP activity by different cofactors or protein domains.
4. Screen the mutant protein of either GAPs or GTPases for activity and substrate specificity.
5. Identification of GAP inhibitors in HTS (high throughput screen) format. Please inquire for significant discounts on large quantities of any reagents in this kit.
Kit Components :
This kit contains sufficient reagents for approximately 20 assays with each small G-protein, and 12 reactions with the positive control GAP protein. The assay can be adapted to a 384 well format which will allow approximately three fold the number of assays. Upon receipt the kit can be stored at 4°C.
Reaction Buffer (2x) One bottle, lyophilized. Contains 10 ml of buffer.
p50 RhoGAP domain Two tubes, lyophilized. Contains 50 µg each of purified p50 RhoGAP domain protein.
His-RhoA protein One tube, lyophilized. Contains 100 µg of purified His tagged RhoA protein.
His-Rac1 protein One tube, lyophilized. Contains 100 µg of purified His tagged Rac1 protein.
His-Cdc42 protein One tube, lyophilized. Contains 100 µg of purified His tagged Cdc42 protein.
His-Ras p21protein One tube, lyophilized. Contains 100 µg of purified His tagged Ras p21 protein.
GTP stock One tube, lyophilized. Contains 100 µl of 100 mM GTP.
CytoPhos Reagent One bottle, liquid. Contains 70 ml of reagent.
96 well plate One half area 96 well plate.
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