Recombinant E. coli Glycerol Kinase Protein, CF
R&D Systems | Catalog # 7849-GK
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Key Product Details
- R&D Systems E. coli-derived Recombinant E. coli Glycerol Kinase Protein (7849-GK)
- Quality control testing to verify active proteins with lot specific assays by in-house scientists
- All R&D Systems proteins are covered with a 100% guarantee
Source
E. coli
Accession Number
Applications
Enzyme Activity
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Product Specifications
Source
E. coli-derived e. coli Glycerol Kinase protein
Met1-Glu502, with a C-terminal 6-His tag
Met1-Glu502, with a C-terminal 6-His tag
Purity
>75%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
N-terminal Sequence Analysis
Thr2
Predicted Molecular Mass
57 kDa
SDS-PAGE
52-55 kDa, reducing conditions
Activity
Measured by its ability to transfer phosphate from ATP to glycerol.
The specific activity is >1,500 pmol/min/μg, as measured under the described conditions.
The specific activity is >1,500 pmol/min/μg, as measured under the described conditions.
Reviewed Applications
Read 1 review rated 4 using 7849-GK in the following applications:
Formulation, Preparation, and Storage
7849-GK
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol, Brij-35 and DTT. |
| Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Background: Glycerol Kinase
References
- Pettigrew, D.W. et al. (1988) J. Biol. Chem. 263: 135.
- Voegele, R.T. et al. (1993) J. Bacteriol. 175:1087.
- Applebee, M.K. et al. (2011) J. Biol. Chem. 286: 23150.
- Hurley, J.H. et al. (1993) Science 259:673.
- Feese, M.D. et al. (1998) Structure 6:1407.
- Wu, Z.L. (2011) PLoS ONE 6(8): e23172.
Alternate Names
GK, GK1, GKD, glpK, Glycerokinase
Gene Symbol
GK
UniProt
Additional Glycerol Kinase Products
Product Documents for Recombinant E. coli Glycerol Kinase Protein, CF
Certificate of Analysis
To download a Certificate of Analysis, please enter a lot or batch number in the search box below.
Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant E. coli Glycerol Kinase Protein, CF
Coomassie is a registered trademark of Imperial Chemical Industries Ltd.
For research use only
Related Research Areas
Customer Reviews for Recombinant E. coli Glycerol Kinase Protein, CF (1)
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Application: Enzymatic activity in vitroVerified Customer | Posted 03/14/2017
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Protocols
View specific protocols for Recombinant E. coli Glycerol Kinase Protein, CF (7849-GK):
Materials
- Assay Buffer (1X): 25 mM HEPES, 150 mM NaCl, 10 mM MgCl2, 10 mM CaCl2, pH 7.0
- Recombinant E. coli Glycerol Kinase (rE.coli glpK) (Catalog # 7849-GK)
- Donor Substrate: Adenosine triphosphate (provided in kit)
- Acceptor Substrate: Glycerol (J.T. Baker, Catalog # 4043), 100 mM in deionized water
- Universal Kinase Activity Kit (Catalog # EA004)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute 1 mM Phosphate Standard by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 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 Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Create a reaction mixture containing 0.5 mM ATP and 12.5 mM Glycerol in Assay Buffer.
- Dilute rE.coli glpK to 2.5 µg/mL in Assay Buffer.
- Dilute Coupling Enzyme to 10 µg/mL in Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Load 20 µL of the 2.5 µg/mL rE.coli glpK into the plate. Include a Control containing 20 µL of Assay Buffer.
- Load 10 µL of the 10 µg/mL Coupling Phosphatase into the wells containing enzyme and blanks.
- Load 20 µL of the reaction mixture into the wells containing enzyme and blanks to start the reactions.
- Incubate sealed plate at room temperature for 10 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 coupling rate** |
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.
**Experimentally derived (6). Under the assay conditions, the coupling rate is 0.475.
Per Reaction:
- rE.coli glpK: 0.05 µg
- ATP: 10 nmol
- Glycerol: 250 nmol
- Coupling Phosphatase 4: 0.1 µg
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