Product Uses
- Study of R-Ras exchange activity with different GEFs.
- Identification of R-Ras exchange factors (GEFs)
- Positive control for GEF studies.
- Biochemical characterization of R-Ras protein interactions
- Western blot standard
Materials
The wild-type human R-Ras protein has been produced in a bacterial expression system. The recombinant protein contains six histidine residues at its amino terminus (His-tag). The molecular weight of 6xHis tagged R-Ras is approximately 25 kDa and is supplied as a white lyophilized powder.
Storage
Before reconstitution, briefly centrifuge to collect the product at the bottom of the tube. The protein should be reconstituted to 5 mg/ml with the addition of 20 µl of Milli-Q water (100 µg size). When reconstituted, the protein will be in the following buffer: 50 mM Tris pH 7.5, 50 mM NaCl, 0.5 mM MgCl2, 5% (w/v) sucrose and 1% (w/v) dextran. In order to maintain high biological activity of the protein it is strongly recommended that the protein solution be supplemented with DTT to 1 mM final concentration, aliquoted into "experiment sized" amounts, snap frozen in liquid nitrogen and stored a -70°C. The protein is stable for six months if stored at -70°C. The protein should not be exposed to repeated freeze-thaw cycles. The lyophilized protein is stable at 4°C desiccated (<10% humidity) for one year.
Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% polyacrylamide gradient gel. His tagged R-Ras protein was determined to be >90% pure. (see Figure 1).
Biological Activity Assay
The biological activity of R-Ras can be determined from the ability of the SOS1 exchange domain (SOS1-ExD) to catalyze the exchange of GDP for GTP on R-Ras. A standard biological assay for monitoring the biological activity of R-Ras is an exchange assay utilizing the 2X Exchange Buffer from the Rho-GEF exchange assay kit (Cat.# BK100) and the human SOS1 GEF domain (Cat.# CS-SOS1).
