| Assay Method Information | |
| | Inhibition Assay |
| Description: | Buffer-I: 50 mM Tris, pH 7.5 150 mM NaCl (optional) 1 mM MgCl2 1 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 | |
|---|---|
| If you find an error in this entry please send us an E-mail | |