Recombinant Human SULT2B1 Protein, CF

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Summary
Reactivity HuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Human SULT2B1 Protein, CF Summary

Details of Functionality
Measured by its ability to transfer sulfate from PAPS to pregnenolone. The specific activity is >10 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived human Cytosolic Sulfotransferase 2B1/SULT2B1 protein
Met1-Glu311, with a C-terminal 6-His tag
Accession #
N-terminal Sequence
Met1
Protein/Peptide Type
Recombinant Enzymes
Gene
SULT2B1
Purity
>80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
36 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
34-37 kDa, reducing conditions
Publications
Read Publication using
6174-ST in the following applications:

Packaging, Storage & Formulations

Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol, Brij and DTT.
Purity
>80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Assay Procedure
  • Assay Buffer: 50 mM Tris, 15 mM MgCl2, pH 7.5 (supplied in 10X form in kit)
  • Recombinant Human Cytosolic Sulfotransferase 2B1/SULT2B1 (rhSULT2B1) (Catalog # 6174-ST)
  • Pregnenolone (Sigma, Catalog # P9129), 50 mM stock in DMSO
  • Dimethylsulfoxide (DMSO)
  • 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
  1. Dilute Coupling Phosphatase 3 to 0.1 mg/mL in Assay Buffer.
  2. Dilute pregnenolone to 5 mM in DMSO.
  3. Dilute PAPS to 1 mM in Assay Buffer.
  4. Prepare reaction mixture by combining 50 µL of 0.1 mg/mL Coupling Phosphatase 3, 20 µL of 5 mM pregnenolone, 100 µL of 1 mM PAPS, and 80 µL of Assay Buffer. This is sufficient to assay 9 wells.
  5. Dilute rhSULT2B1 to 80 µg/mL in Assay Buffer.
  6. 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.
  7. Continue 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.
  8. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  9. Load 25 µL of the 80 µg/mL rhSULT2B1 into the plate. Include a Control containing 25 µL of Assay Buffer.
  10. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  11. Seal plate and incubate at 37 °C for 30 minutes.
  12. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  13. Add 100 µL of deionized water to all wells. Mix briefly.
  14. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  15. Read plate at 620 nm (absorbance) in endpoint mode.
  16. 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:
  • rhSULT2B1: 2.0 µg
  • Coupling Phosphatase 3: 0.5 µg
  • Pregnenolone: 0.2 mM
  • PAPS: 0.2 mM
    • Notes

      Coomassie is a registered trademark of Imperial Chemical Industries Ltd.


      This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

      Alternate Names for Recombinant Human SULT2B1 Protein, CF

      • Alcohol sulfotransferase
      • Cytosolic Sulfotransferase 2B1
      • EC 2.8.2
      • EC 2.8.2.2
      • HSST2
      • HSST2sulfotransferase family cytosolic 2B member 1
      • Hydroxysteroid sulfotransferase 2
      • ST2B1
      • Sulfotransferase 2B1
      • sulfotransferase family, cytosolic, 2B, member 1
      • SULT2B1

      Background

      Cytosolic sulfotransferases catalyze the sulfonation of many hormones, neurotransmitters, drugs, and xenobiotic compounds. They are distinct from Golgi resident sulfotransferases by the absence of transmembrane domains and are located in the cytoplasm (1, 2). Compared to other cytosolic sulfotransferase, SULT2B1 is unique in that it contains a Pro‑rich region between amino acid 305 to 364 that may be related to the thermostability and nuclear translocation of the enzyme (3). The gene for SULT2B1 encodes two isoforms, SULT2B1a and SULT2B1b, that differ only at their amino termini (4). Our recombinant enzyme corresponds to SULT2B1b. SULT2B1b preferentially sulfonates cholesterol and is less active on pregnenolone, DHEA and androstenediol; whereas SULT2B1a preferentially sulfonates pregnenolone and has minimal activity on cholesterol (5). The enzyme activity was determined using a phosphatase-coupled method (6).
      1. Falany, C. N. (1997) FASEB J. 11:206.
      2. Gamage, N. U. et al. (2006) Toxicol. Sci. 90:5.
      3. He, D. and Falany, C. N. (2006) Drug Metab. Dispos. 34:1749.
      4. Her, C. et al. (1998) Genomics. 53:284.
      5. Fuda, H. et al. (2002) J. Biol. Chem. 277:36361.
      6. Prather, B. et al. (2012) Anal. Biochem. 423:86.

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      Publications for Cytosolic Sulfotransferase 2B1/SULT2B1 (6174-ST)(1)

      We have publications tested in 1 confirmed species: Human.

      We have publications tested in 1 application: Bioassay.


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      Bioinformatics

      Gene Symbol SULT2B1
      Uniprot