Recombinant Human ST3GAL2 Protein, CF

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

Recombinant Human ST3GAL2 Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer Neu5Ac from CMP-Neu5Ac to bovine B1-3 galactosyl-N-acetyl galactosamine. The specific activity is >250 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human ST3 beta-Gal alpha-2,3-Sialyltransferase 2/ST3GAL2 protein
  Pro52-Gln350, with a C-terminal 6-His tag
• Accession #: NP_008858
• N-terminal Sequence: Gly56
• Protein/Peptide Type: Recombinant Enzymes
• Gene: ST3GAL2
• Purity: >95%, 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: 35 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: 30-40 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Sialyltransferase Activity Kit (Catalog # EA002)
  • Assay Buffer: 50 mM Tris, 5 mM MnCl₂, pH 7.5 (components supplied in kit)
  • Recombinant Human beta -Gal alpha -2,3-Sialyltransferase 2/ST3GAL2 (rhST3GAL2) (Catalog # 7275-GT)
  • CMP-Sialic Acid (Sigma, Catalog # C8271), 10 mM stock in deionized water
  • beta 1-3 Galactosyl-N-acetyl galactosamine (Dextra, Catalog # GN213), 20 mM stock in deionized water
  • 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. This is the first point of the standard curve.
  2. 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.
  3. Prepare a reaction mixture containing 0.4 mM CMP-Sialic Acid, 1 mM beta 1-3 Galactosyl-N-acetyl galactosamine and 2 µg/mL Coupling Phosphate 2 in Assay Buffer.
  4. Dilute rhST3GAL2 to 2 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 2 µg/mL rhST3GAL2 into the plate. Include a control containing 25 µL of Assay Buffer.
  7. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  8. Seal plate and incubate at 37 °C for 20 minutes.
  9. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  10. Add 100 µL of deionized water to all wells. Mix briefly.
  11. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  12. Read plate at 620 nm (absorbance) in endpoint mode.
  13. 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:
  • rhST3GAL2: 0.050 µg
  • Coupling Phosphatase 2: 0.050 µg
  • CMP-Sialic Acid: 0.2 mM
  • beta 1-3 Galactosyl-N-acetyl galactosamine: 0.5 mM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human ST3GAL2 Protein, CF

• Alpha 2,3-ST 2
• Beta-galactoside alpha-2,3-sialyltransferase 2
• beta-galactoside alpha-2,3-sialytransferase
• CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 2
• EC 2.4.99.4
• EC 2.4.99.7
• Gal-beta-1,3-GalNAc-alpha-2,3-sialyltransferase
• Gal-NAc6S
• sialyltransferase 4B (beta-galactosidase alpha-2,3-sialytransferase)
• Sialyltransferase 4B
• SIAT4B
• SIAT4-B
• ST3 beta-galactoside alpha-2,3-sialyltransferase 2
• ST3GAL2
• ST3GalA.2
• ST3GalA.2ST3Gal II
• ST3GALII

Background

Sialyltransferases add sialic acid to glycoproteins or glycosphingolipids and play important roles in many biological processes including immune recognition, pathogen infection, and cell adhesion (1). Similar to ST3GAL1 (2), ST3GAL2 is a type II membrane protein localized in the trans-Golgi network that can transfer sialic acid to the Gal beta 1-3GalNAc structure to form NeuAc alpha 2-3Gal beta 1-3GalNAc found in terminal carbohydrate groups of particular glycoproteins, oligosaccharides and glycolipids (3). Unlike the ubiquitously expressed ST3GAL1 (4), ST3GAL2 is mainly expressed in heart, liver, skeletal muscle and various lymphoid tissues but not in brain and kidney (3). ST3GAL2 is responsible for the synthesis of monosialosyl globopentaosylceramide (MSGb5), also known as stage-specific embryonic antigen-4 (SSEA4) (5), a cell surface marker for testicular germ cell carcinoma, renal cell carcinoma, and mesenchymal stem cells (6). The enzymatic activity of recombinant human ST3GAL2 was determined using a phosphatase-coupled glycosyltransferase assay (7).

1. Varki, A. (1999) Glycobiology 2:25.
2. Jeanneau, C. et al. (2004) J. Biol. Chem. 279:13461.
3. Giordanengo, V. et al. (1997) Eur. J. Biochem. 247:558.
4. Kitagawa, H. and Paulson, J.C. (1994) J. Biol. Chem. 269:17872.
5. Saito, S. et al. (2003) J. Biol. Chem. 278:26474.
6. Suila, H. et al. (2011) J. Mol. Cell Biol. 3:99.
7. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Bioinformatics

• Gene Symbol: ST3GAL2
• Uniprot:
  • NP_008858()