Product Uses

• Study of G12C K-Ras4B exchange activity with different GEFs

• Identification of G12C K-Ras4B exchange factors (GEFs)

• Positive control for GEF studies

• Biochemical characterization of G12C K-Ras4B protein interactions

• Western blot standard

Materials

The G12C (glycine to cysteine at amino acid position 12) mutant human 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 G12C K-Ras4B is approximately 25 kDa and it is supplied as a white lyophilized powder. 

Storage and Reconstitution

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 ice cold nanopure 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 at -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 G12C K-Ras4B protein was determined to be >85% pure. (see Figure 1).