>80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
<1.0 EU per 1 μg of the protein by the LAL method.
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.
34-37 kDa, reducing conditions
Read Publication using 6174-ST in the following applications:
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.
rhSULT2B1: 2.0 µg
Coupling Phosphatase 3: 0.5 µg
Pregnenolone: 0.2 mM
PAPS: 0.2 mM
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
Cytosolic Sulfotransferase 2B1
HSST2sulfotransferase family cytosolic 2B member 1
Hydroxysteroid sulfotransferase 2
sulfotransferase family, cytosolic, 2B, member 1
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.
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