Recombinant Human POGLUT1 Protein, CF
Recombinant Human POGLUT1 Protein, CF Summary
Product Specifications
Met1-Leu388, with a C-terminal 6-His tag
Accession # Q8NBL1
Analysis
Product Datasheets
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.
6437-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.
|
Assay Procedure
- Assay Buffer: 50 mM HEPES, 10 mM MnCl2, 5 mM CaCl2, pH 7.5
- Recombinant Human O‑Glucosyltransferase 1/POGLUT (rhPOGLUT1) (Catalog # 6437-GT)
- Coupling Enzyme: Recombinant Human CD39L3/ENTPD3 (rhCD39L3/ENTPD3) (Catalog # 4400-EN)
- Substrate: UDP-Glucose (Calbiochem, Catalog # 670120), 10 mM stock in 25% ethanol
- Malachite Green Phosphate Detection Kit (Catalog # DY996)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute UDP-Glucose to 800 μM in Assay Buffer.
- Dilute rhCD39L3 to 4 μg/mL in Assay Buffer.
- Prepare reaction mixture by combining equal volumes of 800 μM UDP-Glucose and 4 μg/mL rhCD39L3.
- Dilute rhPOGLUT1 to 40 µg/mL in Assay Buffer.
- Dilute 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 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.
- 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 40 µg/mL rhPOGLUT1 into the plate. Include a substrate blank containing 25 µL of Assay Buffer.
- Add 25 µL of reaction mixture to the wells, excluding the standard curve and curve blank.
- Cover the plate with a plate sealer and incubate at 37 ºC for 2 hours.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
- Add 100 µL of deionized water to all wells.
- 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 Substrate Blank.
Per Reaction:- rhPOGLUT1: 1 µg
- rhCD39L3: 50 ng
- Substrate: 200 µM
Reconstitution Calculator
Background: Protein O-Glucosyltransferase 1/POGLUT1
O-Glucosyltransferase1 (POGLUT1) is a homologue of Rumi from Drosophila, an endoplasmic reticulum (ER)-retaining glucosyltransferase, that adds glucose to serine residues within the consensus sequence of C1‑X‑S‑X‑P‑C2 in Notch EGF repeats, thereby regulating cell-fate decisions (1). It is also known as CAP10-like protein (2) and KTELC1 due to the highly conserved CAP10 domain and the presence of an ER-retaining motif, KTEL, at the C‑terminus. The human gene is reported to be involved in the pathogenesis of both acute myelogenous and T‑acute lymphoblastic leukemias (3). Recently, POGLUT1 has been demonstrated in a phosphatase-coupled glycosyltransferase assay to have hydrolase activity on UDP-Glc (4).
- Acar, M. et al. (2008) Cell 132:247.
- Teng, Y. et al. (2006) Gene 371:7
- Wang, Y. et al. (2010) Genet Test Mol Biomarkers 14:127.
- Wu, Z.L. et al. (2010) Glycobiology, in press.
Product Specific Notices
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