详细说明
- Purity>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
- Endotoxin Level<0.1 EU per 1 μg of the protein by the LAL method.
- ActivityMeasured by its ability to transfer GlcNAc from UDP-GlcNAc to Recombinant Drosophila Notch EGF20 (Catalog # ). The specific activity is >0.7 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com
- SourceMouse myeloma cell line, NS0-derived His23-Lys523, with C-terminal 6-His tag
- Accession #
- N-terminal Sequence
AnalysisHis23 - Predicted Molecular Mass59 kDa
- SDS-PAGE57-61 kDa, reducing conditions
8116-GT | | |
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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- Assay Buffer: 25 mM Tris, 10 mM MgCl2, 10 mM CaCl2, pH 7.5
- Recombinant Mouse EOGT (rmEOGT) (Catalog # 8116-GT)
- Recombinant Drosophila Notch EGF20 (Notch peptide) (Catalog # )
- UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% EtOH
- Glycosyltransferase Activity Kit (Catalog # )
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
- Prepare standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
- Dilute UDP-GlcNac to 10 mM in Assay Buffer.
- Dilute Coupling Phosphatase I to 20 ng/µL in Assay Buffer.
- Prepare reaction mixture containing 0.8 mM UDP-GlcNAc, 0.2 mg/mL Notch Peptide and 4 ng/µL Coupling Phosphatase I in Assay Buffer.
- Dilute rmEOGT to 20 ng/µL 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 25 µL of the 20 ng/µL rmEOGT into the plate. Include a Control containing 25 µL of Assay Buffer.
- Add 25 µL of reaction mixture to the wells, excluding the standard curve and curve blank.
- Incubate sealed plate at 37 ºC for 4 hours.
- 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) |
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.
Per Reaction:- rmEOGT: 0.5 µg
- Coupling Phosphatase I: 0.1 µg
- Notch Peptide: 5 µg
- UDP-GlcNAc: 0.4 mM
- References:
- Hart, G. W. et al. (2007) Nature, 446:1017.
- Slawson, C., et al. (2006) J. Cell Biochem. 97:71.
- Comer, F. I. and Hart, G. W. (2001), Biochemistry 40:7845.
- Love, D. C. and Hanover, J. A. (2005), Sci. STKE 2005: re13.
- Sakaidani, Y. et al. (2011) Nat. Commun. 2:583.
- Haltiwanger, R. S. et al. (1990) J. Biol. Chem. 265:2563.
- Sakaidani, Y. et al. (2012), Biochem. Biophys. Res. Commun. 419:14.
- Gao, Y. et al. (2001), J. Biol. Chem. 276: 9838-45.
- Muller, R. et al. (2013) PLoS One 8:e62835.
- Cohen, I. et al. (2013) Eur. J. Hum. Genet. Online publication ahead of print.
- Shaheen, R. et al. (2013) Am. J. Hum. Genet. 92 :598.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
- Long Name:EGF Domain-specific O-linked N-acetylglucosamine (GlcNAc) Transferase
- Entrez Gene IDs:285203 (Human); 101351 (Mouse)
- Alternate Names:AER61 glycosyltransferase; AER61; C3orf64; chromosome 3 open reading frame 64; EC 2.4; EOGT; FLJ13078; FLJ33770; FLJ41219; MGC34132