Recombinant Human ST3GAL6 Fc Chimera Protein, CF

2 μg/lane of Recombinant Human ST3GAL6 Fc Chimera Protein (Catalog # 10591-GT) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 67-81 kDa.

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

Recombinant Human ST3GAL6 Fc Chimera Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer Neu5Ac from CMP-Neu5Ac to N-Acetyllactosamine. The specific activity is >50 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human ST3GAL6 protein
  Asn40-Asp331, with N-termininal Fc
• Accession #: Q9Y274.1
• Protein/Peptide Type: Recombinant Enzymes
• 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: 60.1 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: 67-81 kDa, under 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:
  • Assay Buffer: 25 mM Tris, 150 mM NaCl, 5 mM CaCl₂, 10 mM MnCl₂, pH 7.5
  • Sialyltransferase Activity Kit (Catalog # EA002)
  • Recombinant Human ST3GAL6 (rhST3GAL6) (Catalog # 10591-GT)
  • CMP-Sialic Acid (Calbiochem, Catalog # 233264), 2.5 mM stock in deionized water
  • N-Acetyllactosamine (Dextra Laboratories, Catalog # GN204), 50 mM stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. 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 Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  2. Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using 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, 8 mM N-Acetyllactosamine and 4 µg/mL Coupling Phosphatase 2 (supplied in kit) in Assay Buffer.
  4. Dilute rhST3GAL6 to 20 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 20 µg/mL rhST3GAL6 into empty wells of the same plate as the curve. 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 30 minutes.
  9. Add 30 µL of the Malachite Green Reagent A (supplied in kit) to all wells. Mix briefly.
  10. Add 100 µL of deionized water to all wells.
  11. Add 30 µL of the Malachite Green Reagent B (supplied in kit) 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:
  • rhST3GAL6: 0.5 µg
  • Coupling Phosphatase 2: 0.1 µg
  • CMP-Sialic Acid: 0.2 mM
  • N-Acetyllactosamine: 4 mM

Notes

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

Alternate Names for Recombinant Human ST3GAL6 Fc Chimera Protein, CF

• alpha2,3-sialyltransferase
• CMP-NeuAc:beta-galactoside alpha-2,3-sialyltransferase VI
• EC 2.4.99
• EC 2.4.99.-
• EC 2.4.99.9
• Sialyltransferase 10
• SIAT10
• ST3 beta-galactoside alpha-2,3-sialyltransferase 6
• ST3Gal VI
• ST3GAL6
• ST3GALVI
• ST3GALVIsialyltransferase 10 (alpha-2,3-sialyltransferase VI)
• type 2 lactosamine alpha-2,3-sialyltransferase

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). ST3GAL6 is an alpha 2,3-sialyltransferase and forms alpha 2-3 linked sialic acid on Gal-beta 1,4-GlcNAc structure on glycoproteins and glycolipids (2). This enzyme has high specificity for some gangliosides and glycoproteins and may contribute to the formation of sialyl Lewis x (sLex), a carbohydrate important for cell-to-cell recognition and a blood group antigen (3). Study on various alpha 2,3-sialyltransferases indicate that ST3GAL6 selectively sialylates EGFR therefore affects ERK and AKT signal transduction pathways and positively correlates to cell proliferation and colony formation, which is in sharp contrast to ST3GAL4 that selectively sialylates beta 1 integrin (4).

1. Varki, A. (1999) Glycobiology 2:25.
2. Okajima, T. et al. (1999) J Biol Chem. 274:11479.
3. Glavey, S. et al. (2014) Blood 124:1765.
4. Qi, F. et al. (2020) FASEB J. 34:881.

Publications for ST3GAL6 (10591-GT)(1)

Publication using 10591-GT

• ZL Wu, JM Ertelt Fluorescent glycan fingerprinting of SARS2 spike proteins Scientific Reports, 2021-10-14;11(1):20428. 2021-10-14 [PMID: 34650101] (Bioassay, Human)

Bioinformatics

• Uniprot:
  • Q9Y274.1()