Kras4B Protein, Human (G12C, His)

Materials
ATPase/GTPase Assay Kit: Includes assay buffer, Reagent and Pi Standard (1 mM)
Guanosine-5'-triphosphate disodium salt (GTP, HY-W010737): Prepare a 6.67 mM GTP solution in assay buffer.
Kras4B Protein, Human (G12C, His) (HY-P70232)

Procedure
Preparation of Standard Curve
1. Dilute Pi Standard with ddH₂O to concentrations of 100, 50, 30, 15, 0 µM.
2. Add 40 µL of each standard concentration to the wells.
3. Add 200 µL of Reagent to each well and incubate at room temperature (22-25°C) for 30 minutes. 4. Measure the OD value at 630 nm using a microplate reader.
5. Plot the measured OD values on the y-axis and the Pi Standard concentrations on the x-axis to obtain the standard curve equation.

Protein Activity Assay
1. Dilute GTP to 6.67 mM in assay buffer.
2. Dilute Human Kras4B protein to 10 µg/mL in assay buffer.
3. Add 20 µL of Assay Buffer to the wells of a 96-well plate, followed by 10 µL of 10 µg/mL Human Kras4B solution, 10 µL of 6.67 mM GTP solution, and 200 µL of Reagent. For blank wells, add 30 µL of assay buffer, 10 µL of 4 mM GTP solution, and 200 µL of Reagent.
4. Incubate at room temperature (22-25°C) for 30 minutes.
5. Measure the OD value at 630 nm using a microplate reader.
6. Calculate specific activity: