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Description
KRAS(Q61H) His Tag Protein, HumanProduct Specification Species Human Synonyms K Ras 2, Ki Ras, c K ras, c Ki ras, GTPase KRas, KRAS2, RASK2 Accession P01116 2 Amino Acid Sequence Thr2 Cys185(Q61H) with His Tag at the C Terminus Expression System E. coli Molecular Weight 15 25kDa (Reducing) Purity 90% by SDS PAGE Conjugation Unconjugated Tag His Tag Physical Appearance Liquid Storage Buffer 50mM Tris, 200mM NaCl, 20% Glycerol, 1mM DTT, PH7. 5 Stability & Storage Stable for 12 months
Product Specification
| Species | Human |
| Synonyms | K-Ras 2, Ki-Ras, c-K-ras, c-Ki-ras, GTPase KRas, KRAS2, RASK2 |
| Accession | P01116-2 |
| Amino Acid Sequence | Thr2-Cys185(Q61H) with His Tag at the C-Terminus |
| Expression System | E.coli |
| Molecular Weight | 15-25kDa (Reducing) |
| Purity | ＞90% by SDS-PAGE |
| Conjugation | Unconjugated |
| Tag | His Tag |
| Physical Appearance | Liquid |
| Storage Buffer | 50mM Tris, 200mM NaCl, 20% Glycerol, 1mM DTT, PH7.5 |
| Stability & Storage | Stable for 12 months upon stored at -80℃ from the date of receipt. And avoid repeated freeze-thaws cycles. |
| Reference | 1. Gremer L, Merbitz-Zahradnik T, Dvorsky R, Cirstea IC, Kratz CP, Zenker M, Wittinghofer A, Ahmadian MR. Germline KRAS mutations cause aberrant biochemical and physical properties leading to developmental disorders. Hum Mutat. 2011 Jan;32(1):33-43. |
Background
KRAS is a small GTPase protein located at the inner cell membrane, functioning as a molecular switch that regulates cell proliferation and survival signals. The Q61H mutation resides in the Switch II region, impairing GTPase activity and locking the protein in a constitutively GTP-bound "active" state, leading to aberrant activation of MAPK/PI3K pathways. This mutation is commonly found in pancreatic, colorectal, and non-small cell lung cancers. Compared to KRAS G12 mutations, Q61H exhibits resistance to SHP2 inhibitors and is associated with worse prognosis. Clinical detection of this mutation provides crucial guidance for targeted therapy selection and prognostic evaluation.
Protocol
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(Q61H) 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
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