Download Recombinant Human Heparan Sulfate 3-O

Recombinant Human Heparan Sulfate
3-O-Sulfotransferase 1/HS3ST1
Catalog Number: 5968-ST
DESCRIPTION
Source Chinese Hamster Ovary cell line, CHO­derived Arg21­His307, with an N­terminal 6­His tag Accession # O14792 N­terminal Sequence His Analysis Predicted Molecular 35 kDa Mass SPECIFICATIONS SDS­PAGE 38­50 kDa, reducing conditions Activity Measured by its ability to transfer sulfate from PAPS to heparan sulfate. The specific activity is >30 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com. Endotoxin Level <1.0 EU per 1 μg of the protein by the LAL method. Purity >90%, by SDS­PAGE under reducing conditions and visualized by silver stain. Formulation
Supplied as a 0.2 μm filtered solution in Tris­HCl and NaCl. See Certificate of Analysis for details.
Activity Assay Protocol Materials Assay l
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Universal Sulfotransferase Activity Kit (Catalog # EA003) 10X Assay Buffer (supplied in kit): 500 mM Tris, 150 mM MgCl2, pH 7.5 Recombinant Human Heparan Sulfate3­O­Sulfotransferase 1/HS3ST1 (rhHS3ST1) (Catalog # 5968­ST) Donor Substrate: PAP32S (3'­Phosphoadenosine­5'­Phosphosulfate) (Catalog # ES019) Acceptor Substrate: Heparan Sulfate (Celsus Labs, Catalog # HO­3105), 50 mg/mL stock in deionized water 96­well Clear Plate (Catalog # DY990) Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent Prepare 1X Assay Buffer by combining 10X stock and diluting 10 fold with deionized water. Dilute 1 mM Phosphate Standard by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve. Continue standard curve by performing six one­half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer. Prepare a reaction mixture containing 0.4 mM PAP32S and 4 mg/mL Heparin Sulfate in 1X Assay Buffer. Dilute Coupling Phosphatase 3 (supplied in kit) to 50 µg/mL in 1X Assay Buffer. Dilute rhHS3ST1 to 100 µg/mL in 1X Assay Buffer. Load 15 µL of the 100 µg/mL rhHS3ST1 into empty wells of the same plate as the curve. Include a control containing 15 µL of Assay Buffer. Add 10 µL of 50 µg/mL Coupling Phosphatase 3 to wells containing enzyme and control, excluding the standard curve. Add 25 µL of reaction mixture to the wells, excluding the standard curve. Seal plate and incubate at 37 ºC 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 4­parameter fitting and adjusted for Control. Final Assay Conditions Per Reaction: l
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rhHS3ST1: 1.5 µg Coupling Phosphatase 3: 0.5 µg Heparan Sulfate: 100 µg PAP32S: 0.2 mM PREPARATION AND STORAGE 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 freeze­thaw cycles.
l 6 months from date of receipt, ­70 °C as supplied. l 3 months, ­70 °C under sterile conditions after opening. Rev. 10/12/2015 Page 1 of 2 Recombinant Human Heparan Sulfate
3-O-Sulfotransferase 1/HS3ST1
Catalog Number: 5968-ST
BACKGROUND
Heparan sulfate is a highly sulfated polysaccharide that can be found on cell surface and within extracellular matrix. It is typically 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 detached from the protein core and 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 can be sulfated at 2­O position by heparan sulfate 2­
O sulfotransferase (HS2ST). The N­acetylglucosamine residues can be sulfated at N, 3­O, and 6­O positions by N­deacetylase/N­sulfotransferases (NDSTs), heparan sulfate 3­O sulfotransferases (HS3STs) and heparan sulfate 6­O sulfotransferases (HS6STs) respectively. There are seven HS3STs in the human genome (3, 4). HS3ST1 is a rate­limiting enzyme for generating an antithrombin­binding pentasaccharide epitope on heparan sulfate and heparin (5, 6). Unlike other sulfotransferases that have signal­anchor domains and are type II membrane integral proteins in Golgi apparatus, HS3ST1 lacks a transmembrane domain and is likely to be an intraluminal enzyme (7, 8). The enzyme activity of the recombinant HS3ST1 is measured using a phosphatase­coupled assay (9). References:
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Bernfield, M. et al. (1999) Annu. Rev. Biochem. 68:729. Esko, J.D. and Selleck, S.B. (2002) Annu. Rev. Biochem. 71:435. Shworak, N.W. et al. (1999) J. Biol. Chem. 274:5170. Xu, D. et al. (2005) Biochem. J. 386:451. Rosenberg, R.D. et al. (1997) J. Clin. Invest. 100:S67. Esko, J.D and Lindahl, U. (2001) J. Clin. Invest. 108:169. Shworak, N.W. et al. (1997) J. Biol. Chem. 272:28008. Liu, J. et al. (1999) J. Biol. Chem. 274:5158. Prather, B. et al. (2012) Anal.Biochem.423:86 Rev. 10/12/2015 Page 2 of 2