K-Ras4B Mutant Protein: R135A (human recombinant with 6xHis-tag)
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The wild-type human G12D+I36N K-Ras4B 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 G12D+I36N K-Ras4B is approximately 25 kDa and is supplied as a white lyophilized powder.
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.
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% polyacrylamide gradient gel. His tagged G12D+I36N K-Ras4B protein was determined to be >90% pure. (see Figure 1).
Biological Activity Assay
The biological activity of G12D+I36N K-Ras4B can be determined from the ability of the SOS1 exchange domain (SOS1-ExD) to catalyze the exchange of GDP for GTP on G12D+I36N K-Ras4B. A standard biological assay for monitoring the biological activity of G12D+I36N K-Ras4B 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).
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