| Assay Method Information | |
| | Ras GTP Binding Domain Inhibition Assay |
| Description: | The following method was developed as specific assay for KRas G12D mutant protein.Buffer-I:50 mM Tris, pH 7.5150 mM NaCl (optional)1 mM MgCl21 mM DTT.KRas G12D mutant protein was expressed as a His-tagged protein. Purified His-KRas G12D protein was diluted in buffer-I to a final concentration of 3-10 μg/ml.200 μl of the diluted His-KRas G12D protein was added to a nickel coated 96 well plate and incubated overnight at 4° C.The next day, wells were washed 3× in 200 μl of Buffer-I.Then 200 μl of Buffer-I were added to each well in the presence of 1% DMSO.Tested compounds were added to the protein-coated wells at a concentration of 20 μM, and incubated for 3 hours at room temperature. While performing IC50 measurements a serial dilution of all tested concentrations was prepared.Then 22 μl of Cy3-GTP or Cy5-GTP was added to each well. The labeled GTP was incubated for 45 min. at room temperature.Following GTP incubation, wells were washed 3× in Buffer-I, and 200 μl of Buffer-I were added to each well.Following washes, the amount of bound labeled-GTP was measured with an Eppendorf AF2200 plate reader. |
| Affinity data for this assay | |
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