详细说明
- Purity>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
- Endotoxin Level<1.0 EU per 1 μg of the protein by the LAL method.
- ActivityMeasured by its ability to transfer phosphate from ATP to GMP. The specific activity is >7,500 pmol/min/μg, as measured under the described conditions.
- SourceE. coli-derived Ser2-Ala197, with an N-terminal Met and 6-His tag
- Accession #
- N-terminal Sequence
AnalysisMet - Predicted Molecular Mass23 kDa
- SDS-PAGE24 kDa, reducing conditions
9267-GU | | |
Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl. | ||
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. | ||
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
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- Universal Kinase Activity Kit (Catalog # )
- 10X Assay Buffer (supplied in kit): 250 mM HEPES, 1.5 M NaCl, 100 mM MgCl2, 100 mM CaCl2, pH 7.0
- Recombinant Human Guanylate Kinase (rhGUK-1) (Catalog # 9267-GU)
- Guanosine 5'-monophosphate (GMP) (Sigma, Catalog # G8377), 10 mM stock in deionized water
- 96-well Clear Plate (Catalog # )
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Prepare 1X Assay Buffer by diluting 10X stock 10 fold with deionized water.
- Dilute 1 mM Phosphate Standard by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
- Continue standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmoles per well.
- Prepare a reaction mixture containing 0.2 mM ATP (supplied in kit) and 0.2 mM GMP in 1X Assay Buffer.
- Dilute rhGUK-1 to 0.0667 µg/mL 1X Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
- Load 15 µL of the 0.0667 µg/mL rhGUK-1 into empty wells of the same plate as the curve. Include a Control containing 15 µL of 1X Assay Buffer.
- Add 25 µL of reaction mixture to all wells, excluding standard curve.
- Seal plate and incubate at room temperature for 30 minutes.
- Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL 1X Assay Buffer.
- Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to wells containing enzyme and control, excluding the standard curve.
- Seal plate and incubate at room temperature for 5 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 100 µL of deionized water to all wells. Mix briefly.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = | Phosphate released* (nmol) x (1000 pmol/nmol) |
Incubation time (min)** x amount of enzyme (µg) x 2*** |
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.
**Incubation time is 35 minutes according to the above protocol.
***Both ADP and GDP can be digested by Coupling Phosphatase 4 and release one unit of phosphate.
Per Reaction:
- rhGUK-1: 0.001 µg
- Coupling Phosphatase 4: 0.1 µg
- ATP: 0.1 mM
- GMP: 0.1 mM
- References:
- Brady, W.A. et al. (1996) J. Bio.Chem. 271:16734.
- Fitzgibbon, J. et al. (1996) FEBS Letters 385:185.
- Da Rocha, A.A. et al. (2006) Pituitary 9:83.
- Wu, Z.L. (2011) PLoS ONE 6:e23172.
- Entrez Gene IDs:2987 (Human); 14923 (Mouse); 303179 (Rat)
- Alternate Names:ATP:GMP Phosphotransferase; Deoxyguanylate Kinase; EC 2.7.4.8; FLJ42686; FLJ43710; GMK; GMP Kinase; Guanosine monophosphate Kinase; guanylate kinase 1; Guanylate Kinase; GUK1