Recombinant Human SULT1C4 Protein, CF
Recombinant Human SULT1C4 Protein, CF Summary
Ala2-Phe302, with an N-terminal Met and 6-His tag
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, Brij-35 and DTT.|
|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.
- Assay Buffer: 25 mM Tris, 15 mM MgCl2, pH 7.5 (Supplied in 10X form in kit)
- Recombinant Human Cytosolic Sulfotransferase 1C4/SULT1C4 (rhSULT1C4) (Catalog # 7095-ST)
- 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) (Catalog # ES019)
- 1-Naphthol (Sigma, Catalog # N1000), 10 mM in deionized water
- Universal Sulfotransferase Activity Kit (Catalog # EA003)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute 1 mM Phosphate Standard provided by the Universal Sulfotransferase Kit 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.
- Dilute Coupling Phosphatase 3 to 0.125 mg/mL in Assay Buffer.
- Prepare reaction mixture containing 0.4 mM PAPS, 1.12 mM 1-Naphthol, and 20 μg/mL Coupling Phosphatase 3.
- Dilute rhSULT1C4 to 6 µg/mL in Assay Buffer.
- 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 6 µg/mL rhSULT1C4 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 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:
- rhSULT1C4: 0.15 µg
- PAPS: 0.2 mM
- 1-Naphthol: 0.56 mM
- Coupling Phosphatase 3: 0.5 µg
Background: Cytosolic Sulfotransferase 1C4/SULT1C4
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). SULT1C4, originally designated as SULT1C2 (3), is expressed at higher levels in fetal lung and kidney and at lower levels in fetal heart. The endogenous substrate for this enzyme is unknown. It shows activity towards p‑nitrophenol and the carcinogenic compound, N‑hydroxy-2-acetylamino-fluorene (N-OH-2AAF) (3, 4).
- Falany, C. N. (1997) FASEB J. 11:206.
- Gamage, N. U. et al. (2006) Toxicol. Sci. 90:5.
- Sakakibara, Y. et al. (1998) J. Biol. Chem. 273:33929.
- Yoshinari, K. et al. (2001) J. Biochem. Mol. Toxicol. 15:67.
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Sulfotransferase Assays and Substrates
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