Recombinant Human NDST1 Protein, CF
Recombinant Human NDST1 Protein, CF Summary
Product Specifications
Ser79-Arg882, with a C-terminal 6-His tag
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.
5967-ST
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, Brij-35 and Glycerol. |
| 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.
|
Assay Procedure
- Universal Sulfotransferase Activity Kit (Catalog # EA003)
- 10X Assay Buffer (supplied in kit): 500 mM Tris, 150 mM MgCl2, pH 7.5
- Recombinant human N‑Deacetylase/N‑Sulfotransferase 1/NDST1 (rhNDST1) (Catalog # 5967-ST)
- PAPS (3'-Phosphoadenosine-5'-Phosphosulfate) (Catalog # ES019)
- Heparan Sulfate (Celsus Labs, Catalog # HO3105), 50 mg/mL stock in deionized water
- 96-well Clear Plate (Catalog # DY990)
- 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 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.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
- Prepare a reaction mixture containing 0.4 mM PAPS, 2 mg/mL Heparin Sulfate, and 20 µg/mL Coupling Phosphatase 3 in 1X Assay Buffer.
- Dilute rhNDST1 to 2 µg/mL in 1X Assay Buffer.
- Load 25 µL of the 2 µg/mL rhNDST1 into empty wells of the same plate as the curve. Include a control containing 25 µL of Assay Buffer.
- Add 25 µL of reaction mixture to the wells, excluding the standard curve.
- Seal plate and incubate at 37 °C for 20 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) |
*Derived from the phosphate standard curve using linear fitting and adjusted for Control.
Per Reaction:- rhNDST1: 0.05 µg
- Coupling Phosphatase 3: 0.5 µg
- Heparan Sulfate: 50 ug
- PAPS: 0.2 mM
Reconstitution Calculator
Background: N-Deacetylase/N-Sulfotransferase 1/NDST1
Heparan sulfate is a highly sulfated polysaccharide that can be found on the cell surface and within extracellular matrix. It is typically covalently attached to the protein core of proteoglycans, such as syndecans and glypicans (1, 2). Heparin, on the other hand, is a highly sulfated version of heparan sulfate with no protein core and is predominantly found in mast cells. Both heparin and heparan sulfate contain disaccharide repeats of uronic acid and N‑acetylglucosamine and are modified by the same sulfotransferases (1, 2). The uronic acid residues can be sulfated at the 2-O position by heparan sulfate 2-O sulfotransferase (HS2ST). The N‑acetylglucosamine residues can be sulfated at the N, 3-O, and 6-O positions by N-deacetylase/N-sulfotransferases (NDSTs), heparan sulfate 3‑O sulfotransferases and heparan sulfate 6‑O sulfotransferases, respectively. All these enzymes are Golgi resident proteins. NDST-mediated modifications are believed to occur before modifications by other sulfotransferases (3). There are four NDSTs in the human genome and all are dual enzymes with both deacetylase and sulfotransferase domains (4, 5). NDST1 has both strong deacetylase and sulfotransferase activities. NDST1 deficiency causes developmental defects such as cerebral hypoplasia and craniofacial defects (6). The activity of this recombinant NDST1 is determined using a phosphatase coupled assay (7).
- Bernfield, M. et al. (1999) Annu. Rev. Biochem. 68:729.
- Esko, J. D. and Selleck, S. B. (2002) Annu. Rev. Biochem. 71:435.
- Carlsson, P. et al. (2008) J. Biol. Chem. 283:20008.
- Aikawa, J. and Esko, J. D. (1999) J. Biol. Chem. 274:2690.
- Aikawa, J. et al. (2001) J. Biol. Chem. 276:5876.
- Grobe, K. et al. (2005) Development 132:3777.
- Prather, B. et al. (2012) Anal. Biochem. 423:86.
Citations for Recombinant Human NDST1 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 2
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Structural and functional study of D-glucuronyl C5-epimerase.
Authors: Qin Y, Ke J, Gu X, Fang J, Wang W, Cong Q, Li J, Tan J, Brunzelle J, Zhang C, Jiang Y, Melcher K, Li J, Xu H, Ding K
J Biol Chem, 2015-01-07;290(8):4620-30.
Species: Zebrafish
Sample Types: Protein
Applications: Bioassay -
Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases.
Authors: Wu ZL, Prather B, Ethen CM
Glycobiology, 2010-12-17;21(5):625-33.
Species: Bovine, Porcine
Sample Types: Heparin, Protein
Applications: Enzyme Assay
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