Recombinant Human ST6GAL2 Protein, CF Summary
Glu243-Ser529, with a C-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, NaCl and Glycerol.|
|Shipping||The product is shipped with polar packs. 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)
- 10X Assay Buffer (supplied in kit): 250 mM Tris, pH 7.5
- MnCl2 (supplied in kit): 100 mM
- Recombinant Human ST6 Gal Sialyltransferase 2/ST6GAL2 (rhST6GAL2) (Catalog # 8330-GT)
- Recombinant C. perfringens Neuraminidase (rCpNA) (Catalog # 5080-NM)
- CMP-Sialic Acid (Sigma, Catalog # C8271), 10 mM stock in deionized water
- Fetuin (Sigma, Catalog # F3385), 50 mg/mL in deionized water
- 96-well Clear Plate (R&D Systems, Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Prepare 1X Assay Buffer containing 10 mM MnCl2 by combining 10X stocks and diluting 10-fold 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 1X 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 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Prepare reaction mixture containing 0.4 mM CMP-Sialic Acid, 10 mg/mL Fetuin, 8 µg/mL rCpNA and 4 µg/mL Coupling Phosphatase 2 in 1X Assay Buffer.
- Dilute rhST6Gal2 to 80 µg/mL in 1X Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
- Load 25 µL of the 80 µg/mL rhST6Gal2 into the plate. Include a control containing 25 µL of 1X Assay Buffer.
- Add 25 µL of reaction mixture to the wells, excluding the standard curve.
- Seal plate and incubate at room temperature overnight.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 100 µL of deionized water to all wells. Mix briefly.
- 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 fitting and adjusted for Control.Per Reaction:
- rhST6Gal2: 2 µg
- Coupling Phosphatase 2: 0.1 µg
- rCpNA: 0.2 µg
- Fetuin: 0.25 mg
- CMP-Sialic Acid: 200 µM
Background: ST6 Gal Sialyltransferase 2/ST6GAL2
Sialic acid attached to glycoproteins or glycolipids plays important roles in various biological processes such as immune recognition, pathogen infection, and cell adhesion (1). Sialyltransferases are key enzymes in the biosynthesis of these sialoglycoconjugates. Human has two beta -galactoside alpha 2,6-sialyltransferases, ST6GAL1 and ST6GAL1, with 48.9% amino acid sequence identity (2). While ST6GAL1 is expressed in almost all human tissues, ST6GAL2 shows a restricted tissue-specific pattern of expression, mostly in embryonic and adult brain (3). While ST6GAL1 has broad substrate specificity, ST6GAL2 catalyzes 2,6-sialylation of non-reducing end Gal beta 1-4GlcNAc disaccharide on oligosaccharides and exhibits relatively low or no activities toward glycoproteins and glycolipids (4). Bovine ST6GAL2 prefers the disaccharide GalNAc beta 1-4GlcNAc than the disaccharide Gal beta 1-4GlcNAc (5).
- Varki, A. et al. (1999) Essentials of Glycobiology, ColdSpring Harbor Laboratory Press, pp195.
- Takashima, S. et al. (2002) J. Biol. Chem. 277:45719.
- Lehoux, S. et al. (2009) Glycoconj. J. 27:99.
- Takashima, S. et al. (2003) J. Biochem. 134:287.
- Laporte, B. et al. (2009) Glycobiology 19:1082.
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