Recombinant Human SULT2A1 Protein, CF
Recombinant Human SULT2A1 Protein, CF Summary
Product Specifications
Ser2-Glu285, with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
5828-ST
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl 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 Procedure
- Assay Buffer: 50 mM Tris, 15 mM MgCl2, pH 7.5 (supplied in 10X form in kit)
- Recombinant Human Cytosolic Sulfotransferase 2A1/SULT2A1 (rhSULT2A1) (Catalog # 5828-ST)
- 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) (Catalog # ES019)
- T-Dehydroandrosterone (DHEA) (Sigma, Catalog # D4000), 1 mM in 50% DMSO, 50% deionized water
- 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.
- Prepare reaction mixture by combining 140 µL of 1 mM PAPS, 70 µL of 1 mM DHEA, 70 µL of 0.1 mg/mL Coupling Phosphatase 3, and 70 µL of Assay Buffer.
- Dilute rhSULT2A1 to 40 µg/mL in Assay Buffer.
- Dilute 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 5.0 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 rhSULT2A1 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.
- Cover the plate with parafilm or a plate sealer 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:- rhSULT2A1: 1.0 µg
- PAPS: 10000 pmoles (0.2 mM)
- DHEA: 0.1 mM
- Coupling Phosphatase 3: 0.5 µg
Reconstitution Calculator
Background: Cytosolic Sulfotransferase 2A1/SULT2A1
Cytosolic sulfotransferases are a family of Phase II drug-metabolizing enzymes that catalyze the sulfation of many endogenous and xenobiotic substrates (1‑3). They have important roles in the metabolism of many endogenous compounds including steroids, bile acids, thyroid hormones and monoamine neurotransmitters. They are distributed throughout the body and serve to inactivate and increase water-solubility of xenobiotics and therapeutic drugs. They are distinct from Golgi resident sulfotransferases by lacking N-terminal signal-anchor domains and residing only in the cytoplasm. SULT2A1 is primarily expressed in liver and adrenal tissues where it sulfates steroids and bile acids (4‑6). It is also called DHEA/bile acid sulfotransferase and plays roles in maintaining cholesterol and bile acid homeostasis by increasing cholesterol catabolism and, at the same time, preventing toxicity from bile acid accumulation. The enzymatic activity of the recombinant human SULT2A1 is measured using a phosphatase-coupled assay (7).
- Falany, C. N. (1997) FASEB J. 11:206.
- Gamage, N. U. et al. (2006) Toxicol. Sci. 90:5.
- Allali-Hassani, A. et al. (2007) PLoS Biol. 5:e97.
- Comer, K. A. et al. (1993) Biochem. J. 289:233.
- Otterness, D.M. et al. (1992) Mol. Pharmacol. 41:865.
- Luu-The, V. et al. (1995) DNA Cell Biol. 14: 511.
- Prather, B. et al. (2012) Anal. Biochem. 423:86.
Citations for Recombinant Human SULT2A1 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.
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Citations: Showing 1 - 3
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Characterization of human sulfotransferases catalyzing the formation of p-cresol sulfate and identification of mefenamic acid as a potent metabolism inhibitor and potential therapeutic agent for detoxification
Authors: Y Rong, TKL Kiang
Toxicology and Applied Pharmacology, 2021-04-27;0(0):115553.
Species: Human
Sample Types: Protein
Applications: Bioassay -
LC-MS/MS quantification of sulfotransferases is better than conventional immunogenic methods in determining human liver SULT activities: implication in precision medicine
Authors: C Xie, TM Yan, JM Chen, XY Li, J Zou, LJ Zhu, LL Lu, Y Wang, FY Zhou, ZQ Liu, M Hu
Sci Rep, 2017-06-20;7(1):3858.
Species: Human
Sample Types: Protein
Applications: Enzyme Assay -
A versatile polyacrylamide gel electrophoresis based sulfotransferase assay.
Authors: Wu ZL, Ethen CM, Larson S, Prather B, Jiang W
BMC Biotechnol., 2010-02-10;10(0):11.
Applications: Enzyme Assay
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