Recombinant Mouse CHST7 Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse CHST7 Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer sulfate from PAPS to N-acetyl-D-glucosamine. The specific activity is >25 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived mouse Carbohydrate Sulfotransferase 7/CHST7 protein
  Ser34-Val484, with an N-terminal 6-His tag
  Accession # Q9EP78
• Accession #: Q9EP78
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Chst7
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 52 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 55-60 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer (provided in kit): 50 mM Tris, 15 mM MgCl₂, pH 7.5
  • Recombinant Mouse Carbohydrate Sulfotransferase 7/CHST7 (rmCHST7) (Catalog # 5108‑ST)
  • 3'-Phosphoadenosine-5'-phosphosulfate/PAPS (Catalog # ES019)
  • N-acetyl-alpha -D-glucosamine (GlcNAc) (Calbiochem, Catalog # 1079), 1 M stock in deionized water
  • Universal Sulfotransferase Activity Kit (Catalog # EA003)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute 1 mM Phosphate Standard provided by the Universal Sulfotransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
  2. Prepare 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.
  3. Prepare reaction mixture containing 0.4 mM PAPS, 30 mM GlcNAc, and 20 μg/mL Coupling Phosphatase 3 in Assay Buffer.
  4. Dilute rmCHST7 to 10 µg/mL in Assay Buffer.
  5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  6. Load 25 µL of the 10 µg/mL rmCHST7 into the plate. Include a Control containing 25 µL of Assay Buffer.
  7. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  8. Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.
  9. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  10. Add 100 µL of deionized water to all wells. Mix briefly.
  11. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  12. Read plate at 620 nm (absorbance) in endpoint mode.
  13. 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:
  • rmCHST7: 0.25 μg
  • Coupling phosphatase 3: 0.5 μg
  • PAPS: 0.2 mM
  • GlcNAc: 15 mM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse CHST7 Protein, CF

• C6ST2
• carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 7
• Carbohydrate Sulfotransferase 7
• CHST7
• GlcNAc6ST-4
• GST-5

Background

The CHST family is comprised of 14 genes in both human and mouse. All members of this family are Golgi‑localized type II membrane proteins. Only the luminal and enzymatic domain is expressed in each of our recombinant CHST proteins. These enzymes transfer sulfate (i.e., sulfonate) onto the 6‑O or 4‑O positions of GalNAc, Gal and GlcNAc residues on glycoproteins, proteoglycans and glycolipids (1). This sulfation often creates specific epitopes that can be recognized by extracellular matrix proteins, cell surface receptors and viruses (2). CHST7, also designated as chondroitin 6‑O‑sulfotransferase (C6ST‑2), N‑acetylglucosamine-6‑O‑sulfotransferase (GlcNAc6ST-4) and Gal/GalNAc/GlcNAc 6‑O‑sulfotransferase (GST‑5), was previously shown to act on chondroitin sulfate, mannose‑linked GlcNAc and mucin associated GlcNAc residues (3‑5). The amino acid sequence of mouse CHST7 is 87% identical to that of the human counterpart. The enzymatic activity of the recombinant mouse CHST7 was measured using a phosphatase-coupled assay (6).

1. Hemmerich, S. and Rosen, S. (2000) Glycobiology 10:849.
2. Bowman, K. G. and Bertozzi, C. R. (1999) Chem. Biol. 5:447.
3. Kitagawa, H. et al. (2000) J. Biol. Chem. 275:21075.
4. Uchimura, K. et al. (2000) Biochem. Biophys. Res. Commun. 274:291.
5. Bhakta, S. et al. (2000) J. Biol. Chem. 275:40226.
6. Prather, B. et al. (2012) Anal. Biochem. 423:86.

Bioinformatics

• Gene Symbol: Chst7
• Uniprot:
  • Q9EP78()