Recombinant Human GAL3ST2 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human GAL3ST2 Protein, CF Summary

• Details of Functionality: Measured 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.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Galactose-3-O-sulfotransferase 2/GAL3ST2 protein
  His32-Ala398 with an N-terminal 6-His tag
• Accession #: Q9H3Q3
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: GAL3ST2
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 43 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.
• SDS-PAGE: 55-65 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • 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 # ES019)
  • Acceptor Substrate: alpha -Lactose (Sigma, Catalog # L2643), 0.3 M stock 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
  1. 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.
  2. 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.
  3. Prepare a Substrate mixture of 0.8 mM PAPS and 10 mM alpha -Lactose in Assay Buffer.
  4. Dilute rhGAL3ST2 to 4 µg/mL in Assay Buffer.
  5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  6. Load 25 µL of the 4 µg/mL rhGAL3ST2 into the plate. Include a Control containing 25 µL of Assay Buffer.
  7. Add 25 µL of Substrate mixture to the wells, excluding the standard curve.
  8. Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.
  9. Dilute Coupling Phosphatase 3 (supplied in kit) to 10 µg/mL in Coupling Phosphatase Buffer.
  10. 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.
  11. Cover the plate with a plate sealer and incubate at 37 °C for 10 minutes.
  12. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  13. Add 50 µL of deionized water to all wells. Mix briefly.
  14. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  15. Read plate at 620 nm (absorbance) in endpoint mode.
  16. 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.

  Per Reaction:
  • rhGAL3ST2: 0.1 µg
  • Coupling Phosphatase 3: 0.5 µg
  • PAPS: 20 nmol
  • alpha -Lactose: 250 nmol

Notes

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 GAL3ST2 Protein, CF

• GAL3ST2
• GAL3ST-2
• Galactose-3-O-sulfotransferase 2
• GP3ST

Background

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).

1. Honke, K. et al. (2001) J. Biol. Chem. 276: 267.
2. Ideo, H. et al. (2002) Glycobiology 12:199.
3. Ideo, H. et al. (2011) J. Biol. Chem. 286:11346.
4. Prather, B. et al. (2012) Anal. Biochem. 423:86.

Publications for Galactose-3-O-sulfotransferase 2/GAL3ST2 (7719-ST)(1)

Publication using 7719-ST

• C Quin, SD Vicaretti, NA Mohtarudin, AM Garner, DM Vollman, DL Gibson, WF Zandberg Influence of sulfonated and diet-derived human milk oligosaccharides on the infant microbiome and immune markers J. Biol. Chem., 2020-02-03;0(0):. 2020-02-03 [PMID: 32014993] (Enzyme Assay)

Bioinformatics

• Gene Symbol: GAL3ST2
• Uniprot:
  • Q9H3Q3(Human)