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 heparan sulfate. The specific activity is >40 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .
- SourceChinese Hamster Ovary cell line, CHO-derived Pro28-Trp470, with an N-terminal 6-His tag
- Accession #
- N-terminal Sequence
AnalysisHis - Predicted Molecular Mass53 kDa
- SDS-PAGE70-90 kDa, reducing conditions
5406-ST | | |
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.
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- Universal Sulfotransferase Activity Kit (Catalog # )
- 10X Assay Buffer (supplied in kit): 500 mM Tris, 150 mM MgCl2, pH 7.5
- Recombinant Mouse Heparan Sulfate 6-O-Sulfotransferase 3/HS6ST3 (rhHS6ST3) (Catalog # 5406-ST)
- Donor Substrate: PAP32S (3'-Phosphoadenosine-5'-Phosphosulfate) (Catalog # )
- Acceptor Substrate: Heparan Sulfate (Celsus Labs, Catalog # HO3105), 50 mg/mL stock in deionized water
- 96-well Clear Plate (Catalog # )
- 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 one-half 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 6 mg/mL Heparan Sulfate in 1X Assay Buffer.
- Dilute Coupling Phosphatase 3 (supplied in kit) to 50 µg/mL in 1X Assay Buffer.
- Dilute rmHS6ST3 to 33.34 µg/mL in 1X Assay Buffer.
- Load 15 µL of the 33.34 µg/mL rmHS6ST3 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 4-parameter fitting and adjusted for Control.
Per Reaction:- rmHS6ST3: 0.5 µg
- Coupling Phosphatase 3: 0.5 µg
- Heparan Sulfate: 150 ug
- PAP32S: 0.2 mM
- References:
- 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. (2000) J. Biol. Chem. 275:2859.
- Prather, B. et al. (2012) Anal. Biochem. 423:86.
- Entrez Gene IDs:266722 (Human); 50787 (Mouse)
- Alternate Names:DKFZp761K2315; heparan sulfate 6-O-sulfotransferase 3; HS6ST3