1μg (R: reducing condition, N:non-reducing condition).
KRAS(G12D) His Tag Protein, Human
KRAS(G12D) His Tag Protein, Human
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Product Details
Product Details
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（G12D）with His Tag at the C-Terminus |
| Expression System | E.coli |
| Molecular Weight |
20-25kDa (Reducing) |
| Purity | ＞95% by SDS-PAGE |
| Conjugation | Unconjugated |
| Tag | His Tag |
| Physical Appearance | Liquid |
| Storage Buffer |
20 mM Tris, 150 mM NaCl, 1 mM DTT, PH7.4 ,10% glycerol |
| Reconstitution |
Reconstitute at 0.1-1 mg/ml according to the size in ultrapure water after rapid centrifugation. |
| 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
The KRAS protein (Kirsten rat sarcoma viral oncogene homolog) is a small GTPase protein whose structure comprises a G-domain (responsible for GTP/GDP binding) and a hypervariable region. The G12D mutation refers to the substitution of glycine at position 12 (Gly12) with aspartic acid (Asp), which prevents GAP (GTPase-activating protein) from promoting GTP hydrolysis, thereby locking KRAS in a persistently GTP-bound "active" state. This leads to constitutive activation of downstream signaling pathways including RAF-MEK-ERK and PI3K-AKT, driving cellular proliferation, survival, and metabolic reprogramming. Clinically, KRAS G12D represents one of the most prevalent oncogenic mutations in pancreatic cancer (~40%), colorectal cancer, and lung cancer, and has historically been considered "undruggable." However, recent breakthroughs include the development of non-covalent inhibitors (binding the Switch II pocket via salt bridge formation), elucidation of synergistic tumorigenic mechanisms between PTEN loss and G12D, demonstration of G12D-induced immunosuppressive microenvironment conferring resistance to PD-1/PD-L1 inhibitors, and discovery of G12D-specific stem cell reprogramming in lung adenocarcinoma, offering novel directions for precision-targeted therapies and combination immunotherapeutic strategies.
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(G12D) 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
Experimental method
Experimental Principle: The GTPase Glo™ Assay evaluates KRAS activity by measuring the amount of remaining GTP after GTP hydrolysis in the KRAS reaction.
Experimental Materials
1.KRAS(G12D) 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)
Picture
Picture
SDS-PAGE
