Recombinant Human ST8SIA6 Protein, CF SummaryLearn more about Fluorescent Glycan Labeling and Detection
Asp28-Ala398, with an N-terminal 6-His tag
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: 50 mM MES, 10 mM MnCl2, pH 6.5
- Recombinant Human ST8 alpha-2,8-Sialyltransferase (rhST8SIA6) (Catalog # 9587-GT)
- CMP-Neu5Ac (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 (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- 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 nmol per well.
- Prepare reaction mixture containing 0.5 mM CMP-Neu5Ac, 20 mg/mL Fetuin, and 4 µg/mL coupling phosphatase 2 (supplied in kit) in Assay Buffer.
- Dilute rhST8SIA6 to 20 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 20 µg/mL rhST8SIA6 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.
- Incubate sealed plate at 37 °C 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. 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 or 4-parameter fitting and adjusted for Control.Per Reaction:
- rhST8SIA6: 0.5 µg
- Coupling Phosphatase 2: 0.1 µg
- CMP-Neu5Ac: 250 µM
- Fetuin: 500 µg
Background: ST8 alpha-2,8-Sialyltransferase 6/ST8SIA6
Polysialic acid (PSA), a glycan abundant on the neural cell adhesion molecule (NCAM) during embryonic development, negatively modulates the adhesive properties of NCAM (1). Following birth, PSA expression decreases promptly and becomes restricted to the hippocampus, hypothalamus, and olfactory bulb-areas of the brain that require continuous cell migration and synaptic plasticity (2). Expression of PSA in cancer cells has been suggested to increase tumor invasiveness and promote tumor growth (3). However; the mouse St8sia6 only has alpha -2,8 sialyltransferase activity toward both glycolipids and glycoproteins that has the NeuAc-alpha -2,3(6)-beta -D-Gal sequence at the nonreducing ends of their carbohydrate groups, and forms NeuAc-alpha -2,8-NeuAc structures, but not oligosialic or polysialic acid structures (4). Like most glycosyltransferases, ST8SIA6 may be a Golgi‑resident type II membrane protein. It is expressed in various tissues at low levels but is highly expressed in breast cancer and Dami megakaryocyte cell lines (5). The activity of this enzyme has been measured with a phosphatase-coupled method (6).
- Scheidegger, E.P. et al. (1995) J. Biol. Chem. 270:22685.
- Rutishauser, U. (2008) Nat. Rev. Neurosci. 9:26.
- Seidenfaden, R. et al. (2003) Mol.Cell. Biol. 23:5908.
- Takashima, S. et al. (2002) J. Biol. Chem. 277:24030.
- Teintenier-Lelievre, M. et al. (2005) Biochem. J. 392: 665.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
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