Recombinant Human ST6GAL1 (aa 44-406) Protein, CF

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

Recombinant Human ST6GAL1 (aa 44-406) Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer Neu5Ac from CMP-Neu5Ac to N-Acetyllactosamine. The specific activity is >150 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human ST6 Gal Sialyltransferase 1/ST6GAL1 protein
  Glu44-Cys406 with an N-terminal 6-His tag
• Accession #: P15907
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: ST6GAL1
• 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: 42 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: 40-50 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:
  • Assay Buffer: 25 mM Tris, 150 mM NaCl, 5 mM CaCl₂, 10 mM MnCl₂, pH 7.5 (Tris and MnCl₂ supplied in kit)
  • Recombinant Human Gal Sialyltransferase 1/ST6GAL1 (rhST6GAL1) (Catalog # 7620-GT)
  • CMP-Neu5Ac (Sigma, Catalog # C8271), 10 mM stock in deionized water
  • N-Acetyllactosamine (V-Labs, Catalog # GN204), 50 mM stock in deionized water
  • Sialyltransferase Activity Kit (Catalog # EA002)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • 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.
  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 nmoles per well.
  3. Prepare reaction mixture containing 0.4 mM CMP-Neu5Ac, 2 mM N-Acetyllactosamine, and 4 µg/mL Coupling Phosphatase 2 in Assay Buffer.
  4. Dilute rhST6Gal1 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 the 20 µg/mL rhST6GAL1 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. Cover the plate with parafilm or a plate sealer 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.
  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:
  • rhST6GAL1: 0.5 µg
  • Coupling phosphatase 2: 0.1 µg
  • CMP-Neu5Ac: 0.2 mM
  • N-Acetyllactosamine: 1 mM

Notes

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

Alternate Names for Recombinant Human ST6GAL1 (aa 44-406) Protein, CF

• Alpha 2,6-ST
• B-cell antigen CD75
• beta-galactoside alpha-2,6-sialyltransferase 1
• CD75
• CMP-N-acetylneuraminate beta-galactosamide alpha-2,6-sialyltransferase
• CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,6-sialyltransferase 1
• EC 2.4.99.1
• MGC48859
• sialyltransferase 1 (beta-galactoside alpha-2,6-sialyltransferase)
• sialyltransferase 1 (beta-galactoside alpha-2,6-sialytransferase)
• Sialyltransferase 1
• SIAT1
• SIAT1Alpha 2,6-ST 1
• ST6 beta-galactosamide alpha-2,6-sialyltranferase 1
• ST6Gal I
• ST6GAL1
• ST6GalI
• ST6N

Background

Sialic acid molecules attached to glycoproteins or glycosphingo lipids play important roles in various biological processes such as immune recognition, pathogen infection, and cell adhesion (1).Sialyltransferases are key enzymes that regulate the cellular levels of sialic acid containing molecules. Beta-galactosamide alpha‑2,6‑sialyltransferase 1 encoded by the ST6GAL1 gene is a type II membrane protein localized in the trans-Golgi network and catalyzes 2,6-sialylation of Gal beta 1,4‑GlcNAc structures on N-glycans (2). The enzyme is involved in the generation of the cell surface carbohydrate determinants and differentiation antigens HB6, CD75, and CD76 (3). ST6GAL1 is highly expressed in the liver and also expressed in most other tissues to some extent (4). ST6GAL1 deficiency causes abnormalities in B‑cell immunoreactivity (5). The expression and activity of ST6GAL1 are associated with tumor metastasis in breast (6) and colon (7) cancers. The majority of ST6GAL1 in the liver is cleaved and secreted into the serum (8) and may be used as a biomarker for hepatitis diseases (9). Using a phosphatase-coupled sialyltransferase assay, the K_m for the donor substrate CMP-NeuAc was determined to be around 0.5 mM (10).

1. Varki, A. et al. (1999) Essentials of Glycobiology, Cold Spring Harbor Laboratory Press, pp195.
2. Weinstein, J. et al. (1987) J. Biol. Chem. 262:17735.
3. Bast, B.J. et al. (1992) J. Cell Biol. 116:423.
4. Kitagawa, H. and J.C. Paulson (1994) J. Biol. Chem. 269:17872.
5. Hennet, T. et al. (1998) Proc. Natl. Acad. Sci. USA 95:4504.
6. Recchi, M.A. et al. (1998) Cancer Res. 58:4066.
7. Dall’Olio, F. et al. (2001) Eur. J. Biochem. 268:5876.
8. Weinstein, J. et al. (1987) J. Biol. Chem. 262:17735.
9. Kitazume, S. et al. (2009) Glycobiology 19:479.
10.  Wu, Z.L. et al. (2011) Glycobiology 21:727.

Publications for ST6 Gal Sialyltransferase 1/ST6GAL1/CD75 (7620-GT)(5)

Publications using 7620-GT

• Chen, WS;Concio, CAP;Chang, TT;Chen, CL;Perez, SJLP;Li, WS; Optimizing ST6GAL1 inhibition and selectivity using lithocholic acid-amino acid conjugates for antimetastatic and antiangiogenic agent development Bioorganic chemistry 2025-03-22 [PMID: 40174529] (Bioassay, N/A)
• T Jaroentome, YH Kwon, Y Liu, O Young, R Bhawal, JD Wilson, M Li, DG Chapla, KW Moremen, MC Jewett, D Mizrachi, MP DeLisa A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans Nature Communications, 2022-10-24;13(1):6325. 2022-10-24 [PMID: 36280670] (Bioassay, Human)
• 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)
• Wu Z, Person A, Anderson M, Burroughs B, Tatge T, Khatri K, Zou Y, Wang L, Geders T, Zaia J, Sackstein R Imaging specific cellular glycan structures using glycosyltransferases via click chemistry. Glycobiology, 2018-02-01;0(0):. 2018-02-01 [PMID: 29186441] (Click Chemistry, Human, Mouse)
• Mark B Jones B-cell-independent sialylation of IgG Proc Natl Acad Sci USA, 2016-06-14;113(26):7207-12. 2016-06-14 [PMID: 27303031] (Enzyme Assay, Mouse)

Bioinformatics

• Gene Symbol: ST6GAL1
• Uniprot:
  • P15907()