Recombinant Human ST3GAL1 Protein, CF

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

Recombinant Human ST3GAL1 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 >1000 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human ST3 beta-Gal alpha-2,3-Sialyltransferase 1/ST3GAL1 protein
  Arg57-Arg340, with an N-terminal HA (YPYDVPDYA) tag
• Accession #: Q11201
• N-terminal Sequence: Tyr (of HA tag)
• Protein/Peptide Type: Recombinant Enzymes
• Gene: ST3GAL1
• Purity: >85%, 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: 33.4 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: 35-45 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: >85%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Sialyltransferase Activity Kit (Catalog # EA002)
  • 10X Assay Buffer: 250 mM Tris, pH 7.5
  • MnCl₂ (supplied in kit): 100 mM
  • Recombinant Human beta -Gal alpha -2,3-Sialyltransferase/ST3GAL1 (rhST3GAL1) (Catalog # 6905-GT)
  • CMP-Neu5Ac (Sigma, Catalog # C8271), 10 mM stock in deionized water
  • beta 1-3 Galactosyl-N-acetyl galactosamine (V-labs, Catalog # GN213), 50 mM stock in deionized water
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare 1X Assay Buffer containing 10 mM MnCl₂ by combining equal volumes of 10X Assay Buffer and 100 mM MnCl₂ and diluting 5-fold with deionized water.
  2. Dilute Phosphate Standard 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 nmoles per well.
  4. Prepare reaction mixture containing 0.4 mM CMP-Neu5Ac, 1 mM beta 1-3 Galactosyl-N-acetyl galactosamine, 4 μg/mL Coupling Phosphatase 2 in 1X Assay Buffer.
  5. Dilute rhST3GAL1 to 1 µg/mL in 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 1 µg/mL rhST3GAL1 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 37 °C for 20 minutes.
  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) = (Adjusted 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:
  • rhST3GAL1: 0.025 μg
  • Coupling Phosphatase 2: 0.1 μg
  • CMP-Neu5Ac: 200 µM
  • 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 ST3GAL1 Protein, CF

• Alpha 2,3-ST 1
• CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 1
• DKFZp666E036
• DKFZp779K2051
• EC 2.4.99
• EC 2.4.99.4
• Gal-beta-1,3-GalNAc-alpha-2,3-sialyltransferase
• Gal-NAc6S
• sialyltransferase 4A (beta-galactosidase alpha-2,3-sialytransferase)
• sialyltransferase 4A (beta-galactoside alpha-2,3-sialyltransferase)
• sialyltransferase 4A (beta-galactoside alpha-2,3-sialytransferase)
• Sialyltransferase 4A
• SIAT4
• SIAT4A
• SIAT4-A
• SIATFL
• SIATFLFLJ36548
• ST3 beta-galactoside alpha-2,3-sialyltransferase 1
• ST3Gal I
• ST3GAL1
• ST3GalA
• ST3GalA.1MGC9183
• ST3GalI
• ST3GalIA
• ST3GalIA1,Beta-galactoside alpha-2,3-sialyltransferase 1
• ST3O
• ST3OST3GalA

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). ST3GAL1 is a type II membrane protein localized in the trans‑Golgi network that transfers sialic acid to galactose residues of core 1 O‑glycans (3Gal beta 1‑3GalNAc) to produce sialyl‑T antigen (NeuAc alpha 2‑3Gal beta 1‑3GalNAc) (2, 3). ST3GAL1 is ubiquitously expressed in most tissues, with highest expression in the placenta, liver and skeletal muscle (4). Over‑expression of ST3GAL1 may be involved in the tumorigenesis of both breast cancer (5, 6) and bladder cancer (7). The enzyme activity of the recombinant human ST3GAL1 was determined using a phosphatase‑coupled glycosyltransferase assay (8).

1. Varki, A. (1999) Glycobiology 2:25.
2. Jeanneau, C. et al. (2004) J. Biol. Chem. 279:13461.
3. Dalziel, M. et al. (2001) J. Biol. Chem. 276:11007.
4. Kitagawa, H. and Paulson, J.C. (1994) J. Biol. Chem. 269:17872.
5. Picco, G. et al. (2010) Glycobiology 20:1241.
6. Burchell, J. et al. (1999) Glycobiology 9:1307.
7. Videira, P.A. et al. (2009) BMC Cancer 9:357.
8. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Publications for ST3 beta-Gal alpha-2,3-Sialyltransferase 1/ST3GAL1/SIAT4A (6905-GT)(1)

Publication using 6905-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

• Gene Symbol: ST3GAL1
• Uniprot:
  • Q11201()