Recombinant Human Heparan Sulfate-6-O-Sulfotransferase-1, CF
Recombinant Human Heparan Sulfate-6-O-Sulfotransferase-1, CF Summary
Pro28-Trp401, with an N-terminal 6-His tag
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl and EDTA.|
|Shipping||The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 50 mM Tris, 15 mM MgCl2, pH 7.5
- Recombinant Human Heparan Sulfate 6‑O‑Sulfotransferase 1/HS6ST1 (rhHS6ST1) (Catalog # 5057-ST)
- Donor Substrate: 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) (Catalog # ES019)
- Acceptor Substrate: Heparan Sulfate (Celsus Labs, Catalog # HO3105), 50 mg/mL stock in deionized water
- Sulfotransferase Activity Kit (Catalog # EA003)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Create Assay Buffer by Diluting 10X Phosphatase Buffer 3 (supplied in kit) to 1X in 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.
- Continue standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
- Prepare a reaction mixture containing 0.4 mM PAPS, 4 mg/mL Heparin Sulfate, and 20 ug/mL Coupling Phosphatase 3 in Assay Buffer. *Note: Incorporate Coupling Phoshphatase 3 into reaction mixture right before the mixture is added to the plate in order to avoid unnecessary hydrolysis of PAPS.
- Dilute rhHS6ST1 to 20 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 25 µL of the 40 µg/mL rhHS6ST1 into the plate. 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.
- 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:
- rhHS6ST1: 0.5 µg
- Coupling Phosphatase 3: 0.5 µg
- Heparan Sulfate: 100 ug (2 mg/mL)
- PAPS: 0.2 mM
Background: Heparan Sulfate 6-O-Sulfotransferase 1/HS6ST1
Heparan Sulfate is a highly sulfated polysaccharide that can be found on cell surface and extracellular matrix. It is usually covalently attached to a protein core as the glycan component of a proteoglycan. Heparan Sulfate interacts with a variety of proteins, such as growth factors, protease inhibitors, cytokines, lipoprotein lipase and viral envelope proteins, thus plays roles from cell growth, cell differentiation, cell motility, blood coagulation, lipid metabolism to viral infection (1, 2). Heparan Sulfate consists of repeating residues of uronic acid and N‑acetylglucosamine. The uronic acid residues can be sulfated at 2-O position by Heparan Sulfate 2‑O‑Sulfotransferase (HS2ST). The N‑acetylglucosamine residues can be sulfated at N-, 3-O, and 6-O positions by N‑deacetylase/N‑sulfotransferases (NDSTs), Heparan Sulfate 3-O and 6-O sulfotransferases (HS3STs and HS6STs) respectively. However, the reactions catalyzed by these sulfotransferases are normally incomplete on the whole chain of Heparan Sulfate. As a result, Heparan Sulfate displays enormous sequence diversity that allows it to interact with a wide spectrum of proteins differently. Among three HS6STs, HS6ST1 is the first to be cloned (3). Mice deficient of the HS6ST1 homologue gene showed embryonic lethality (4).The enzymatic activity of the recombinant human HS6ST1 is measured using a phosphatase-coupled assay (5).
- Bernfield, M. et al. (1999) Annu. Rev. Biochem. 68:729.
- Esko, J. D. and Selleck, S. B. (2002) Annu. Rev. Biochem. 71:435.
- Habuchi, H et al. (1998) J. Biol. Chem. 273:9208.
- Habuchi, H et al. (2007) J. Biol. Chem. 282:15578.
- Prather, B. et al. (2012) Anal. Biochem. 423:86.
Citations for Recombinant Human Heparan Sulfate-6-O-Sulfotransferase-1, CF
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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;290(8):4620-30.
Sample Types: Protein
Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases.
Authors: Wu ZL, Prather B, Ethen CM
Sample Types: Protein
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