Recombinant Human ST3GAL3 His-tag Protein, CF Summary
Lys29-Ile375, with a N-terminal 6-His tag
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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.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris and NaCl.|
|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.
- Sialyltransferase Activity Kit (Catalog # EA002)
- Assay Buffer: 25 mM Tris, 150 mM NaCl, 5 mM CaCl2, 10 mM MnCl2, pH 7.5 (Components supplied in kit)
- Recombinant Human ST3GAL3 (rhST3GAL3) (Catalog # 10554-GT)
- CMP-Sialic Acid (CMP-Neu5Ac) (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
- Prepare Assay Buffer with deionized water.
- 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.
- 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 nmoles per well.
- Prepare reaction mixture containing 0.4 mM CMP-Sialic Acid, 4 mM N-Acetyllactosamine, and 4 µg/mL Coupling Phosphatase 2 (supplied in kit) in Assay Buffer.
- Dilute rhST3GAL3 to 4 µg/mL in Assay Buffer.
- 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 4 µg/mL of rhST3GAL3 into empty wells of the same plate as the curve. Include a Control containing 25 µL of Assay Buffer.
- Add 25 µL of reaction mixture to the wells, excluding the standard curve.
- Seal plate and incubate at 37 ºC for 20 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- 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 Control.Per Reaction:
- rhST3GAL3: 0.1 µg
- Coupling Phosphatase 2: 0.1 µg
- CMP-Sialic Acid: 200 µM
- N-acetyllactosamine: 2 mM
2 μg/lane of Recombinant Human ST3GAL3 His-tag Protein (Catalog # 10554-GT) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 37-43 kDa.
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). ST3GAL3 is an alpha 2,3-sialyltransferase and forms alpha 2-3 linked sialic acid on the Gal residue of the terminal Gal beta 1-4GlcNAc, Gal beta 1-3GlcNAc and Gal beta 1-3GalNAc structures found on glycoproteins and glycolipids (2). ST3GAL3 is involved in the synthesis of both sialyl Lewis x (sLex), and sialyl Lewis a (sLea) epitopes that can serve as the ligands for E- and P-selectins on activated endothelial cells in inflammation (3). The glycan epitopes formed by ST3GAL3 are a prerequisite for attaining and/or maintaining higher cognitive functions (4) and mutations in this gene have been associated with a form of autosomal recessive nonsyndromic cognitive disability as well as infantile epileptic encephalopathy (5). The enzyme is expressed as a type II membrane protein localized in the trans-Golgi apparatus but can also be proteolytically processed to form a soluble form (6). The activity of the recombinant human ST3GAL3 was determined using a phosphatase‑coupled glycosyltransferase assay (7).
- Varki, A. (1999) Glycobiology 2:25.
- Weinstein, J. et al. (1982) J Biol Chem. 257:13845.
- Grahn, A. et al. (2002) Glycoconjugate Journal 19:197.
- Hu, H. et al. (2011) Am J Hum Genet. 89:407.
- Joshi, H.J. et al. (2018) Glycobiology 28: 284.
- Burger, P. C. et al. (1998) Glycobiology 8:245.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
Citation for Recombinant Human ST3GAL3 His-tag Protein, 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
Fluorescent glycan fingerprinting of SARS2 spike proteins
Authors: ZL Wu, JM Ertelt
Scientific Reports, 2021;11(1):20428.
Sample Types: Recombinant Protein
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