Recombinant Human ST6GAL2 Protein, CF

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

Recombinant Human ST6GAL2 Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer Neu5Ac from CMP-Neu5Ac to fetuin of fetal calf serum. The specific activity is >0.45 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human ST6 Gal Sialyltransferase 2/ST6GAL2 protein
  Glu243-Ser529, with a C-terminal 6-His tag
• Accession #: Q96JF0
• N-terminal Sequence: Glu243
• Protein/Peptide Type: Recombinant Enzymes
• Gene: ST6GAL2
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <0.1 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 33 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: 37-43 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, NaCl and Glycerol.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Sialyltransferase Activity Kit (Catalog # EA002)
  • 10X Assay Buffer (supplied in kit): 250 mM Tris, pH 7.5
  • MnCl₂ (supplied in kit): 100 mM
  • Recombinant Human ST6 Gal Sialyltransferase 2/ST6GAL2 (rhST6GAL2) (Catalog # 8330-GT)
  • Recombinant C. perfringens Neuraminidase (rCpNA) (Catalog # 5080-NM)
  • CMP-Sialic Acid (Sigma, Catalog # C8271), 10 mM stock in deionized water
  • Fetuin (Sigma, Catalog # F3385), 50 mg/mL in deionized water
  • 96-well Clear Plate (R&D Systems, Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare 1X Assay Buffer containing 10 mM MnCl₂ by combining 10X stocks and diluting 10-fold with deionized water.
  2. Dilute 1 mM Phosphate Standard provided by the Sialyltransferase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  3. Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  4. Prepare reaction mixture containing 0.4 mM CMP-Sialic Acid, 10 mg/mL Fetuin, 8 µg/mL rCpNA and 4 µg/mL Coupling Phosphatase 2 in 1X Assay Buffer.
  5. Dilute rhST6Gal2 to 80 µg/mL in 1X Assay Buffer.
  6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
  7. Load 25 µL of the 80 µg/mL rhST6Gal2 into the plate. Include a control containing 25 µL of 1X Assay Buffer.
  8. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  9. Seal plate and incubate at room temperature overnight.
  10. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  11. Add 100 µL of deionized water to all wells. Mix briefly.
  12. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  13. Read plate at 620 nm (absorbance) in endpoint mode.
  14. 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 fitting and adjusted for Control.

  Per Reaction:
  • rhST6Gal2: 2 µg
  • Coupling Phosphatase 2: 0.1 µg
  • rCpNA: 0.2 µg
  • Fetuin: 0.25 mg
  • CMP-Sialic Acid: 200 µM

Notes

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

Alternate Names for Recombinant Human ST6GAL2 Protein, CF

• Alpha 2,6-ST 2
• beta-galactoside alpha-2,6-sialyltransferase 2
• beta-galactoside alpha-2,6-sialyltransferase II
• CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,6-sialyltransferase 2
• EC 2.4.99
• EC 2.4.99.1
• FLJ30711
• FLJ37730
• FLJ38334
• hST6Gal II
• KIAA1877
• KIAA1877St6gal2
• sialyltransferase 2 (monosialoganglioside sialyltransferase)
• Sialyltransferase 2
• SIAT2
• ST6 beta-galactosamide alpha-2,6-sialyltranferase 2
• ST6Gal II
• ST6GAL2
• St6GalII

Background

Sialic acid attached to glycoproteins or glycolipids plays important roles in various biological processes such as immune recognition, pathogen infection, and cell adhesion (1). Sialyltransferases are key enzymes in the biosynthesis of these sialoglycoconjugates. Human has two beta -galactoside alpha 2,6-sialyltransferases, ST6GAL1 and ST6GAL1, with 48.9% amino acid sequence identity (2). While ST6GAL1 is expressed in almost all human tissues, ST6GAL2 shows a restricted tissue-specific pattern of expression, mostly in embryonic and adult brain (3). While ST6GAL1 has broad substrate specificity, ST6GAL2 catalyzes 2,6-sialylation of non-reducing end Gal beta 1-4GlcNAc disaccharide on oligosaccharides and exhibits relatively low or no activities toward glycoproteins and glycolipids (4). Bovine ST6GAL2 prefers the disaccharide GalNAc beta 1-4GlcNAc than the disaccharide Gal beta 1-4GlcNAc (5).

1. Varki, A. et al. (1999) Essentials of Glycobiology, ColdSpring Harbor Laboratory Press, pp195.
2. Takashima, S. et al. (2002) J. Biol. Chem. 277:45719.
3. Lehoux, S. et al. (2009) Glycoconj. J. 27:99.
4. Takashima, S. et al. (2003) J. Biochem. 134:287.
5. Laporte, B. et al. (2009) Glycobiology 19:1082.

Bioinformatics

• Gene Symbol: ST6GAL2
• Uniprot:
  • Q96JF0(Human)