Recombinant Human ST6GAL1, CF Summary
Product Specifications
Lys27-Cys406 with an N-terminal 6-His tag
Accession # P15907
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
5924-GT
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. |
| Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 25 mM Tris, 150 mM NaCl, 5 mM CaCl2, 10 mM MnCl2, pH 7.5
- Recombinant Human Gal Sialyltransferase 1/ST6GAL1 (rhST6GAL1) (Catalog # 5924-GT)
- Coupling Enzyme: Recombinant Human 5'‑Nucleotidase/CD73 (rhCD73) (Catalog # 5795-EN)
- CMP-Neu5Ac (Sigma, Catalog # C8271), 10 mM stock in deionized water]
- N-Acetyllactosamine (V-Labs, Catalog # GN204), 50 mM stock in deionized water
- Malachite Green Phosphate Detection Kit (Catalog # DY996)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute CMP-Neu5Ac to 1.2 mM in Assay Buffer.
- Dilute N-Acetyllactosamine to 6 mM in Assay Buffer.
- Dilute rhCD73 to 12 μg/mL in Assay Buffer.
- Prepare reaction mixture by combining equal volumes of diluted CMP-Neu5Ac, N-Acetyllactosamine, and rhCD73.
- Dilute rhST6GAL1 to 20 µg/mL in Assay Buffer.
- Dilute 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of assay buffer for a 100 µM stock. This is the first point of the standard curve.
- 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 5.0 nmol per well.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Load 25 µL of the 20 µg/mL rhST6GAL1 into the plate. Include a substrate blank containing 25 µL of Assay Buffer.
- Add 25 µL of reaction mixture to the wells, excluding the standard curve.
- Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
- Add 100 µL of deionized water to all wells.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- 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 Substrate Blank.
Per Reaction:- rhST6GAL1: 0.5 μg
- rhCD73: 100 ng
- CMP-Neu5Ac: 200 µM
- N-Acetyllactosamine: 1 mM
Reconstitution Calculator
Background: ST6 Gal Sialyltransferase 1/ST6GAL1
Sialic acid molecules attached to glycoproteins or glycosphingolipids 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 HB-6, 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 glycosyltransferase assay, the Km for the donor substrate CMP-NeuAc was determined to be around 0.5 mM (10).
- Varki, A. et al. (1999) Essentials of Glycobiology, Cold Spring Harbor Laboratory Press, pp195.
- Weinstein, J. et al. (1987) J. Biol. Chem. 262:17735.
- Bast, B.J. et al. (1992) J. Cell Biol. 116:423.
- Kitagawa, H. and J.C. Paulson (1994) J. Biol. Chem. 269:17872.
- Hennet, T. et al. (1998) Proc. Natl. Acad. Sci. USA 95:4504.
- Recchi, M.A. et al. (1998) Cancer Res. 58:4066.
- Dall’Olio, F. et al. (2001) Eur. J. Biochem. 268:5876.
- Weinstein, J. et al. (1987) J. Biol. Chem. 262:17735.
- Kitazume, S. et al. (2009) Glycobiology 19:479.
- Wu, Z.L. et al. (2011) Glycobiology in press.
Citation for Recombinant Human ST6GAL1, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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Galectin-1-mediated cell adhesion, invasion and cell death in human anaplastic large cell lymphoma: regulatory roles of cell surface glycans.
Authors: Suzuki O, Abe M
Int J Oncol, 2014-03-04;44(5):1433-42.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay
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