Recombinant Human HS3ST3B1 Protein, CF Summary
Ser54-Asp390 with a C-terminal 6-His tag
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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, NaCl and Brij-35.|
|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 (supplied in 10X form in kit)
- Recombinant Human Heparan Sulfate Glucosamine 3-O-sulfotransferase 3 (rhHS3ST3B1) (Catalog # 7276-ST)
- Heparan Sulfate (Celsus Labs, Catalog # HO3105), 50 mg/mL stock in deionized water
- 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) (Catalog # ES019)
- Universal Sulfotransferase Activity Kit (Catalog # EA003)
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute Coupling Phosphatase 3 to 60 µg/mL in Assay Buffer.
- Dilute heparan sulfate to 8 mg/mL in Assay Buffer.
- Dilute PAPS to 0.4 mM in Assay Buffer.
- Prepare reaction mixture by combining equal volumes of 60 µg/mL Coupling Phosphatase 3, 8 mg/mL heparan sulfate, and 0.4 mM PAPS.
- Dilute rhHS3ST3B1 to 40 µg/mL in Assay Buffer.
- Dilute the 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. This is the first point of the standard curve.
- Continue standard curve preparation 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.
- 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 rhHS3ST3B1 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. 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:
Background: Heparan Sulfate Glucosamine 3-O-Sulfotransferase 3
Heparan sulfate is a highly sulfated polysaccharide found on the cell surface and within the extracellular matrix. It is typically covalently attached to the protein core of proteoglycans, such as syndecans and glypicans. Heparin, on the other hand, is considered to be 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 can be sulfated at the 2‑O position by heparan sulfate 2‑O sulfotransferase (HS2ST). The N‑acetylglucosamine residues can be sulfated at the N, 3‑O, and 6‑O positions by N‑deacetylase/ N‑sulfotransferases (NDSTs), heparan sulfate 3‑O sulfotransferases (HS3STs) and heparan sulfate 6‑O sulfotransferases (HS6STs) respectively. There are seven HS3STs in the human genome (3, 4). HS3ST3 has two forms, HS3ST3A1 and HS3ST3B1, differing only at the N‑terminus. The two HS3STs have the same substrate specificity (5) and similar tissue distribution with a high levels of expression in the liver and spleen (3, 6). HS3ST3 can sulfate IdoUA2S‑GlcNS, IdoUA2S‑GlcNH2 and IdoUA2S‑GlcNS6S and generate tetrasulfated disaccharide units (3, 6). HS3ST3 is involved in generation of a binding receptor to the herpes simplex virus‑1 (HSV‑1) (7). The enzyme activity was determined using a phosphatase-coupled method (8).
- 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.
- Liu, J. et al. (1999) J. Biol. Chem. 274:5185.
- Mochiziki, H. (2008) J. Biol. Chem. 283:31237.
- Moon, A.F. et al. (2004) J. Biol. Chem. 279:45185.
- Prather, B. et al. (2012) Anal. Biochem. 423:86.
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