详细说明
- Purity>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
- Endotoxin Level<1.0 EU per 1 μg of the protein by the LAL method.
- ActivityMeasured by its ability to transfer sulfate from PAPS to alpha -Lactose. The specific activity is >800 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com
- SourceChinese Hamster Ovary cell line, CHO-derived His32-Ala398 with an N-terminal 6-His tag
- Accession #
- N-terminal Sequence
AnalysisHis - Predicted Molecular Mass43 kDa
- SDS-PAGE55-65 kDa, reducing conditions
| 7719-ST | | |
| Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl. | ||
| 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.
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Assay Procedure
Materials
- Assay Buffer: 25 mM MES, 15 mM MgCl2, pH 5.5
- Coupling Phosphatase Buffer: 100 mM Tris, 15 mM MgCl2, pH 8.0
- Recombinant Human Galactose‑3‑O‑sulfotransferase 2/GAL3ST2 (rhGAL3ST2) (Catalog # 7719-ST)
- Donor Substrate: 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) (Catalog # )
- Acceptor Substrate: alpha -Lactose (Sigma, Catalog # L2643), 0.3 M stock in deionized water
- Universal Sulfotransferase Activity Kit (Catalog # )
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- 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.
- Prepare 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.
- Prepare a Substrate mixture of 0.8 mM PAPS and 10 mM alpha -Lactose in Assay Buffer.
- Dilute rhGAL3ST2 to 4 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 4 µg/mL rhGAL3ST2 into the plate. Include a Control containing 25 µL of Assay Buffer.
- Add 25 µL of Substrate mixture to the wells, excluding the standard curve.
- Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.
- Dilute Coupling Phosphatase 3 (supplied in kit) to 10 µg/mL in Coupling Phosphatase Buffer.
- Add 50 µL of 10 µg/mL Coupling Phosphatase 3 to each well, excluding the standard curve. Add Coupling Phosphatase Buffer to the standard curve.
- Cover the plate with a plate sealer and incubate at 37 °C for 10 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 50 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.
**Use the incubation time of the reaction between the Enzyme and Substrate mixture only, exclude additional incubation times.
- rhGAL3ST2: 0.1 µg
- Coupling Phosphatase 3: 0.5 µg
- PAPS: 20 nmol
- alpha -Lactose: 250 nmol
Background: Galactose-3-O-sulfotransferase 2/GAL3ST2
Galactose-3-O-sulfotransferase 2 (GAL3ST2) is a type II Golgi resident transmembrane protein. It transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the C-3 hydroxyl group of nonreducing beta -galactosyl residues present on various glycans, such as N-acetyllactosamine, lactose, lacto-N-tetraose (Lc4), lacto-N-neotetraose (nLc4), type 1 oligosaccharides (Gal beta 1-3GlcNAc-R), type 2 oligosaccharides (Gal beta 1-4GlcNAc-R), and core 1 O-glycans (Gal beta 1-3GalNAc alpha 1-Ser/Thr) (1). Given its broad substrate specificity, GAL3ST2 is capable of radioisotope labeling of non-reducing terminal galactose on glycoproteins or glycolipids with 35S. In addition, it can be used to generate galactin-4 and galactin-8 specific ligands (2, 3). The protein is widely expressed in various tissues (1). The enzymatic activity of the recombinant human GAL3ST2 is measured using a phosphatase-coupled assay (4).
- References:
- Honke, K. et al. (2001) J. Biol. Chem. 276: 267.
- Ideo, H. et al. (2002) Glycobiology 12:199.
- Ideo, H. et al. (2011) J. Biol. Chem. 286:11346.
- Prather, B. et al. (2012) Anal. Biochem. 423:86.
- Entrez Gene IDs:64090 (Human); 381334 (Mouse); 685402 (Rat)
- Alternate Names:GAL3ST2; Galactose-3-O-sulfotransferase 2; GP3ST
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