KRAS(G12C) His Tag Protein, Human

KRAS(G12C) is an oncogenic mutant of the RAS family member KRAS, in which a glycine at position 12 is mutated to cysteine. This mutation disrupts GTP hydrolysis, causing the protein to remain persistently in the GTP-bound "activated state". The G12C mutation introduces a nucleophilic cysteine into the Switch II pocket, providing a binding site for covalent inhibitors (e.g., Sotorasib, Adagrasib), which lock KRAS in the inactive GDP-bound conformation. This mutation constitutively activates downstream pathways such as RAF-MEK-ERK and PI3K-AKT, driving cell proliferation and survival, making it a key driver in non-small cell lung cancer (NSCLC; ~14%), pancreatic cancer (~2%), and colorectal cancer (~3%). Clinically, KRAS(G12C) was once considered an "undruggable" target until the first covalent inhibitor, AMG 510, emerged in 2019, marking a major breakthrough in precision oncology. To date, the FDA has approved Sotorasib and Adagrasib for second-line treatment of KRAS G12C-mutant NSCLC. Combination therapy with EGFR inhibitors has also shown significant efficacy in colorectal cancer; however, acquired resistance remains a clinical challenge.

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.

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.

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