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

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 the addition of 20 ul of ice cold nanopure water (100 ug 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 -70C. The protein is stable for six months if stored at -70C.The protein should not be exposed to repeated freeze-thaw cycles. The lyophilized protein is stable at 4C desiccated (< 10% humidity) for one year.

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The G12D (glycine to aspartic acid 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 G12D K-Ras4B is approximately 25 kDa and it is supplied as a white lyophilized powder.

1. Dilute SOS1-ExD protein (Cat.# CS-GE02) to 1 ?M (0.06 mg/ml) with Dilution Buffer.
2. Dilute G12D K-Ras4B to 40 uM (1.0 mg/ml) with Dilution Buffer.
3. Dissolve lyophilized 2X Exchange Buffer in 5 ml nanopure water and keep at room temperature.
4. Set up the plate reader for kinetic fluorescence measurements (Excitation wavelength at 360 nm and emission wavelength at 440 nm) with readings every 30 seconds for 30 minutes.
5. Add the following components together and mix well by gentle pipetting:
Association/Exchange reaction mix / 96 well black plate
2x Exchange Buffer / 50 ?l
dH₂O / 26 ?l
40 uM?G12D K-Ras4B/ 4 ?l
6. Pipette 20 ?l of 1 ?M SOS1-ExD protein or Dilution Buffer into their respective wells and immediately pipette up and down twice and begin reading the fluorescence.
7. Once the readings are complete and the plate reader file has been saved, the exchange rate can be calculated by reducing the data to Vmax with the software that accompanies the plate reader.

G12D K-Ras4B Protein Purity Determination. A 20 ug sample of recombinant G12D K-Ras4B protein (molecular weight approx. 25 kDa) was separated by electrophoresis in a 4-20% SDS-PAGE system and stained with Coomassie Blue. Protein quantitation was determined using the Precision Red Protein Assay Reagent (Cat. # ADV02). ?SeeBlue molecular weight markers are from Life Technologies Inc.