Assay protocol

Principle: The GTPase Glo™ Assay assesses the activities of KRAS by detecting the amount of GTP remaining after GTP hydrolysis in a KRAS reaction.

Materials

1.KRAS(G12C) His Tag Protein, Human

2.GTPase Glo™ Assay (Promega, Catalog # V7681T)

3.Solid white multi-well plate (384-well plate) (Corning, Catalog #3572)

4.Plate Reader (PerkinElmer)

Produce

1.Prepare a 2X GTP solution containing 10 µM GTP and 2mM DTT in GTPase/GAP Buffer.

2.Dilute the KRAS to 100 µg/mL、80 µg/mL and 60 µg/mL in GTPase/GAP Buffer and dispense 5 µL into each well of a 384 well plate.

3.Initiate the reaction by adding 5 µL of the 2X GTP solution prepared in Step 1 to each well. Include a 2X GTP solution with 5 µL GTPase/GAP Buffer as Blank. The reaction volume is 10 µL.

4.Incubate the reaction at room temperature (22-25℃) for 30 minutes.

5.Gently mix the thawed GTPase Glo™ Reagent, 500X, by inversion; do not vortex. Prepare the required volume of reconstituted GTPase Glo™ Reagent by increasing or decreasing the component volumes provided below.

Sample Name	Amount
GTPase Glo ™ Reagent, 500X	2 μL
ADP, 10 mM	0.5 μL
GTPase Glo ™ Buffer	998 μL
Total volume	1mL

6.Add 10 µL of reconstituted GTPase-Glo™ Reagent to the completed reaction, mix briefly and incubate with shaking for 30 minutes at room temperature (22–25℃).

7.Add 20 µL of Detection Reagent and incubate the plate for 5-10 minutes at room temperature (22–25℃).

8.Read at emission wavelengths of 555 nm (luminescence), respectively in endpoint mode.

9.Calculate specific activity.

Specific Activity (pmol/min/μg) =	(1-Sample OD/BLANK OD)*50pmol
	Incubation time(min) ×amount of enzyme (μg)

Sample OD: Remains of ATP OD

BLANK OD: Added of GTP OD

50pmol: Added of GTP

Incubation time: 30 min

amount of enzyme: 0.5μg, 0.4 μg and 0.3 μg