Recombinant Human SULT2B1 Protein, CF Summary
Met1-Glu311, 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, Glycerol, Brij and DTT.|
|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: 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
- Dilute Coupling Phosphatase 3 to 0.1 mg/mL in Assay Buffer.
- Dilute pregnenolone to 5 mM in DMSO.
- Dilute PAPS to 1 mM in Assay Buffer.
- 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.
- Dilute rhSULT2B1 to 80 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 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 80 µg/mL rhSULT2B1 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 30 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: Cytosolic Sulfotransferase 2B1/SULT2B1
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).
- Falany, C. N. (1997) FASEB J. 11:206.
- Gamage, N. U. et al. (2006) Toxicol. Sci. 90:5.
- He, D. and Falany, C. N. (2006) Drug Metab. Dispos. 34:1749.
- Her, C. et al. (1998) Genomics. 53:284.
- Fuda, H. et al. (2002) J. Biol. Chem. 277:36361.
- 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 SULT2B1 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
Identification of Galeterone and Abiraterone as Inhibitors of Dehydroepiandrosterone Sulfonation Catalyzed by Human Hepatic Cytosol, SULT2A1, SULT2B1b, and SULT1E1
Authors: CKY Yip, S Bansal, SY Wong, AJ Lau
Drug Metab. Dispos., 2018;0(0):.
Sample Types: Recombinant Protein
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