• 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.  

• Activity

  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. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Chinese Hamster Ovary cell line, CHO-derived Ser34-Val484, with an N-terminal 6-His tag Accession # Q9EP78

• Accession #

• N-terminal Sequence    
  Analysis

  His

• Predicted Molecular Mass

  52 kDa

• SDS-PAGE

  55-60 kDa, reducing conditions

Assay Procedure

Materials

• 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 # )

• N-acetyl-alpha -D-glucosamine (GlcNAc)  (Calbiochem, Catalog # 1079), 1 M stock in deionized water

• Universal Sulfotransferase Activity Kit (Catalog # )

• 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:

     *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

Background: Carbohydrate Sulfotransferase 7/CHST7

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).

• References:

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.

• Entrez Gene IDs:

  56548 (Human); 60322 (Mouse); 302302 (Rat)

• Alternate Names:

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