K-Ras4B Mutant Protein: G12D+I36N (human recombinant with 6xHis-tag)

Materials

The wild-type human G12D+D38A 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+D38A K-Ras4B 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 G12D+D38A K-Ras4B protein was determined to be >90% pure. (see Figure 1).

Biological Activity Assay

The biological activity of G12D+D38A 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+D38A K-Ras4B. A standard biological assay for monitoring the biological activity of G12D+D38A 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).