详细说明
- 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.
- ActivityMeasured by its ability to transfer sulfate from PAPS to chondroitin sulfate. The specific activity is >350 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .
- SourceChinese Hamster Ovary cell line, CHO-derived Glu39-Thr472, with an N-terminal 6-His tag
- Accession #
- N-terminal Sequence
AnalysisHis - Predicted Molecular Mass50 kDa
- SDS-PAGE60-80 kDa, reducing conditions
| 5356-ST | | |
| Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl. | ||
| 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.
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Assay Procedure
Materials
- Assay Buffer: 50 mM Tris, 125 mM NaCl, 50 mM MgCl2, pH 7.5
- Recombinant Mouse Carbohydrate Sulfotransferase 3/CHST3 (rmCHST3) (Catalog # 5356-ST)
- Donor Substrate: Adenosine 3’-phosphate 5’-phosphosulfate (PAPS) (Catalog # )
- Acceptor Substrate: Chondroitin Sulfate (Sigma, Catalog # C6737), 50 mg/mL 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
- Dilute 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 5.0 nmol per well.
- Prepare a reaction mixture containing 0.571 mM PAPS, 14.3 mg/mL chondroitin sulfate, and 14.3 μg/mL Coupling Phosphatase 3.
- Dilute rmCHST3 to 4.17 µg/mL in Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Load 15 µL of the 4.17 µg/mL rmCHST3 into the plate. Include a Control containing 15 µL of Assay Buffer.
- Add 35 µL of reaction mixture to the wells, excluding the standard curve.
- Cover the plate with a plate sealer 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 or 4-parameter fitting and adjusted for Control.
Per Reaction:- rmCHST3: 0.0625 μg
- Coupling Phosphatase 3: 0.500 μg
- PAPS: 0.4 mM
- Chondrointin Sulfate: 500 μg
Background: Carbohydrate Sulfotransferase 3/CHST3
The mouse CHST family is comprised of 13 enzymes. 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). CHST3, also known as chondroitin 6-O-sulfotransferase, transfers sulfate to position 6 of GalNAc residues on chondroitin sulfate (3). Chondroitin sulfate constitutes the predominant proteoglycan present in cartilage and is distributed on the surfaces of many cells and extracellular matrices. Loss of CHST3 function in human results in severe chondrodysplasia (4). CHST3 can also sulfate Gal residues of keratan sulfate and Gal residues in sialyl N-acetyllactosamine (sialyl LacNAc) oligosaccharides (5). Mouse CHST3 shares 85% amino acid sequence identity to the human ortholog.
- References:
- Hemmerich, S. and Rosen, S. (2000) Glycobiology 10:849.
- Bowman, K. G. and Bertozzi, C. R. (1999) Chem. Biol. 5:447.
- Uchimura, K. et al. (2002) J. Biol. Chem. 277:1443.
- Thiele, H. et al. (2004) Proc. Natl. Acad. Sci. U. S. A. 101:10155.
- Yusa, A. et al. (2006) J. Biol. Chem. 281: 20393.
- Entrez Gene IDs:9469 (Human); 53374 (Mouse); 84468 (Rat)
- Alternate Names:C6ST1; C6ST-1; C6ST1HSD; C6STEC 2.8.2.17; carbohydrate (chondroitin 6) sulfotransferase 3; Carbohydrate Sulfotransferase 3; Chondroitin 6-O-sulfotransferase 1; Chondroitin 6-sulfotransferase; CHST3; EC 2.8.2; Galactose/N-acetylglucosamine/N-acetylglucosamine 6-O-sulfotransferase 0; GST-0
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