Tiam1 GEF protein (human recomb. DHPH domain, MBP tagged)

On Arrival: 4°C
Before reconstitution, briefly centrifuge to collect the product at the bottom of the tube. The protein should be reconstituted to 5 mg/ml with ice cold nanopure water (20 ul water per 100 ug protein).When reconstituted, the protein will be in the following buffer: 20 mM Tris pH 7.5, 50 mM NaCl, 1 mM MgCl2, 5% (v/v) sucrose and 1% (v/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 at -70C. The protein is stable for six months if stored at -70C. The protein must not be exposed to repeated freeze-thaw cycles. The lyophilized protein is stable at 4C desiccated (< 10% humidity) for one year. Further Ras-GRF1 dilutions should be made in 20 mM Tris pH 7.5, 50 mM NaCl, 1 mM MgCl2.(not supplied).

RasGRF1 is a guanine exchange factor with selectivity for H-, K-, N- and R-Ras, which mediates Ras isoform activation under a variety of conditions and has been associated with multiple diseases (reviewed in 1, 2).
Study inhibitors of RasGRF GTP/GDP exchange activity
Identification of RasGRF Cdc25 domain binding proteins
Study of RasGRF GEF activity with different GTPases

The biological activity of RasGRF1 CDC25 domain can be determined from its ability to catalyze nucleotide exchange on Ras isoforms using the nucleotide exchange assay of Bodipy-GDP for excess GDP or GTP. Ras protein is pre-loaded with Bodipy-FL-GDP by adding excess EDTA e.g. 0.7 mmol EDTA per mmol Mg2+ ions present in the reaction. This sub-stock solution is then used in a dissociation assay format which indicates competition for exchange site with unlabeled nucleotide. The reaction is monitored by fluorescence measurement at 485nm Ex / 535nm Em. Stringent quality control ensures that the exchange rate of Bodipy-GTP or mant-GTP is enhanced at least five fold in the presence of 0.8 uM RasGRF CDC25 domain.
Reagents
1. Recombinant Ras protein (Cat.# RS01hH-Ras, RS02hN-Ras, RS03hK-Ras, RS04hK-Ras-G12V, RS05hR-Ras.
2. Recombinant RasGRF Cdc25 protein domain (Cat.# CS-GE03)
3. Exchange buffer 2 (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 2 mM EDTA, 100 ug/ml BSA, and 0.75 uM Bodipy-FL-GDP), note - make fresh.
4. 50 mM MgCl2 in 20 mM Tris-HCl pH 7.5, 50 mM NaCl.
5. 5 mM GTP in 20 mM Tris-HCl pH 7.5, 50 mM NaCl.

Equipment
1. Fluorescence spectrometer (?ex=485nm, ?em=525nm, both with< 15nm bandwidth)
2. Corning 96-well half area plates (Cat # 3686) or other plate with low protein binding surface.