Recombinant Human HS2ST1 Protein, CF Summary
Met59-Asn356, with an N-terminal 6-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|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.
- Assay Buffer (provided in kit): 50 mM Tris, 15 mM MgCl2, pH 7.5
- Recombinant Human Heparan Sulfate 2-O-Sulfotransferase 1/HS2ST1 (rhHS2ST1) (Catalog # 6335-ST)
- 3'-Phosphoadenosine-5'-phosphosulfate/PAPS (Catalog # ES019)
- Heparan sulfate (Celsus Laboratories, Catalog # HO-03101), 50 mg/mL 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
- 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.
- 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.
- Prepare reaction mixture containing 0.4 mM PAPS, 6 mg/mL Heparan sulfate, and 20 μg/mL Coupling Phosphatase 3 in Assay Buffer.
- Dilute rhHS2ST1 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 20 µg/mL rhHS2ST1 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.
- 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:
- rhHS2ST1: 0.5 μg
- Coupling Phosphatase 3: 0.5 μg
- PAPS: 0.2 mM
- Heparan sulfate: 150 μg
Background: Heparan Sulfate 2-O-Sulfotransferase 1/HS2ST1
Heparan sulfate is a highly sulfated polysaccharide found on the cell surface and within the extracellular matrix. Typically, it is covalently attached to the protein core of proteoglycans, such as syndecans and glypicans. Heparin, on the other hand, can be considered as a highly sulfated version of heparan sulfate that 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 are either glucuronic acid or iduronic acid and maybe sulfated at the 2-O position by heparan sulfate 2‑O sulfotransferase 1 (HS2ST1) (3, 4). HS2ST1 physically interacts in the Golgi apparatus with glucuronyl c5-epimerase (5), which catalyzes the conversion of glucuronic acid to iduronic acid (6). As a consequence, 2-O sulfation predominantly occurs on iduronic acids naturally and overexpression of HS2ST1 alone causes an increase in 2-O sulfation on glucuronic acid (7). The enzyme activity of the recombinant human HS2ST1 was measured using a PAP-specific phosphatase-coupled sulfotransferase assay (8).
- Bernfield, M. et al. (1999) Annu. Rev. Biochem. 68:729.
- Esko, J.D. and Selleck, S.B. (2002) Annu. Rev. Biochem. 71:435.
- Kobayashi, M. et al. (1996) J. Biol. Chem. 271:7645.
- Bullock, S.L. et al. (1998) Genes & Development 12:1894.
- Li, J.P. et al. (2001) J. Biol. Chem. 276:20069.
- Pinhal, M.A.S et al. (2001) Proc. Natl. Acad. Sci. USA. 98:12984.
- Rong, J. et al. (2000) Biochem. J. 346:463.
- Prather, B. et al. (2012) Anal Biochem. 423:86.
Product Specific NoticesCoomassie is a registered trademark of Imperial Chemical Industries Ltd.
Citation for Recombinant Human HS2ST1 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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
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Sulfotransferase Assays and Substrates
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