Recombinant Human NDST1 Protein, CF

R&D Systems | Catalog # 5967-ST

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Key Product Details

  • R&D Systems CHO-derived Recombinant Human NDST1 Protein (5967-ST)
  • 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

CHO

Accession Number

P52848

Applications

Enzyme Activity
View all N-Deacetylase/N-Sulfotransferase 1/NDST1 Proteins and Enzymes »

Product Specifications

Source

Chinese Hamster Ovary cell line, CHO-derived human N-Deacetylase/N-Sulfotransferase 1/NDST1 protein
Ser79-Arg882, with a C-terminal 6-His tag

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Ser79

Predicted Molecular Mass

93 kDa

SDS-PAGE

90-100 kDa, reducing conditions

Activity

Measured by its ability to transfer sulfate from PAPS to heparan sulfate.
The specific activity is >750 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

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.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

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).

References

  1. Bernfield, M. et al. (1999) Annu. Rev. Biochem. 68:729.
  2. Esko, J. D. and Selleck, S. B. (2002) Annu. Rev. Biochem. 71:435.
  3. Carlsson, P. et al. (2008) J. Biol. Chem. 283:20008.
  4. Aikawa, J. and Esko, J. D. (1999) J. Biol. Chem. 274:2690.
  5. Aikawa, J. et al. (2001) J. Biol. Chem. 276:5876.  
  6. Grobe, K. et al. (2005) Development 132:3777.
  7. Prather, B. et al. (2012) Anal. Biochem. 423:86.

Alternate Names

HSNST1, HSST1, NST1

Entrez Gene IDs

3340 (Human); 15531 (Mouse); 29633 (Rat)

Gene Symbol

NDST1

UniProt

P52848

Additional N-Deacetylase/N-Sulfotransferase 1/NDST1 Products

Product Documents for Recombinant Human NDST1 Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human NDST1 Protein, CF

For research use only

Citations for Recombinant Human NDST1 Protein, CF

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Protocols

View specific protocols for Recombinant Human NDST1 Protein, CF (5967-ST):

Materials
  • 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
  1. Prepare 1X Assay Buffer by diluting 10X stock 10 fold with deionized water.
  2. 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.
  3. 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.
  4. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
  5. 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.
  6. Dilute rhNDST1 to 2 µg/mL in 1X Assay Buffer.
  7. 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.
  8. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  9. Seal plate and incubate at 37 °C for 20 minutes.
  10. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  11. Add 100 µL of deionized water to all wells. Mix briefly.
  12. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  13. Read plate at 620 nm (absorbance) in endpoint mode.
  14. 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

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