Recombinant Drosophila Notch EGF20 Protein, CF Summary
Product Specifications
| Leader Sequence SPWPLD |
Notch (Asp791-Asn829) Accession # P07207 |
Linker | 6-His tag | |
| N-terminus | C-terminus | |||
Analysis
Product Datasheets
8117-DN
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
8117-DN
| 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.
|
Assay Procedure
- 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 8117-DN)
- UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% EtOH
- Glycosyltransferase Activity Kit (Catalog # EA001)
- 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
Background: Notch EGF20
Drosophila notch protein is a signaling protein that regulates the differentiation of eye, wing disk, oogenesis, segmental appendages and nervous system through lateral inhibition or induction. It functions as a receptor for membrane-bound ligands Delta and Serrate to regulate cell-fate determination (1). The mature protein is a transmembrane protein with an extracellular domain from amino acid 53 to 1745, which consists of 36 EGF repeats (1). The EGF repeats are modified with O-linked beta -N-acetylglucosamine (O-GlcNAc) by an extracellular O-GlcNAc transferase (EOGT) (2, 3). The EGF repeat 20 expressed in this construct can be used as a substrate for EOGT (recombinant mouse EOGT, R&D catalogue 8116-GT) (2). It may also be used as a substrate for protein O-Glucosyltransferase 1 (POGLUT) (4) (recombinant human POGLUT, R&D catalogue 6437-GT) and Fringe (5).
- Wharton K.A. et al. (1985) Cell 43:567-581.
- Matsuura A. et al. (2008) J. Biol. Chem. 283:35486.
- Sakaidani, Y. et al. (2012) Biochem. Biophys. Res. Commun. 419:14.
- Acar, M. et al. (2008) Cell 132:247.
- Bruckner K. et al. (2000) Nature 406:411.
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