Recombinant Human ST8SIA2 Protein, CF
Recombinant Human ST8SIA2 Protein, CF Summary
Learn more about Fluorescent Glycan Labeling and DetectionProduct Specifications
Asp24-Thr375, with an N-terminal 6-His tag
Analysis
Product Datasheets
6590-GT
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.
6590-GT
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| 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.
|
Assay Procedure
- Assay Buffer: 50 mM MES, 5 mM MgCl2, pH 7.0
- Recombinant Human alpha -2,8‑Sialyltransferase 8B/ST8SIA2 (rhST8SIA2) (Catalog # 6590-GT)
- Donor Substrate: CMP-Sialic Acid (Sigma, Catalog # C8271), 10 mM stock in deionized water
- Acceptor Substrate: Recombinant Human NCAM-1/CD56 (rhCD56) 120 isoform (Catalog # 2408-NC)
- Sialyltransferase Activity Kit (Catalog # EA002)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute CMP-Sialic Acid to 3 mM in Assay Buffer.
- Dilute Coupling Phosphatase 2 to 24 µg/mL in Assay Buffer.
- Dilute rhCD56 to 240 µg/mL in Assay Buffer.
- Prepare reaction mixture by combining 45 µL of 3 mM CMP-Sialic Acid, 45 µL of 24 µg/mL Coupling Phosphatase 2, and 180 µL of 240 µg/mL rhCD56.
- Dilute rhST8SIA2 to 10 µg/mL in Assay Buffer.
- Dilute 1 mM Phosphate Standard 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.
- 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 10 µg/mL rhST8SIA2 into the plate. Include a Control 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 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:- rhST8SIA2: 0.250 μg
- Coupling Phosphatase 2: 100 ng
- rhCD56: 4 µg
- CMP-Sialic Acid: 250 µM
Background: ST8 alpha-2,8-Sialyltransferase 8B/ST8SIA2
Polysialic acid (PSA), abundant on the neural cell adhesion molecule (NCAM) during embryonic development, acts as an anti-adhesive glycan to negatively modulate the adhesive properties of NCAM (1). PSA expression decreases promptly after birth, 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 to promote tumor growth (3). The temporal regulation of PSA is dependent on the expression of two polysialyltransferases, ST8SIA4 and ST8SIA2 (4, 5). ST8SIA2, also known as sialyltransferase X, is mainly expressed during embryonic development (4) and shows strict preference on NCAM (6). The high degree of substrate specificity is achieved through specific enzyme-substrate recognition at both the protein sequence and glycan structure levels (6, 7). Like most glycosyltransferases, ST8SIA2 is a Golgi‑resident type II membrane protein. The activity of this enzyme has been measured with a phosphatase-coupled method (8).
- 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.
- Angata, K. et al. (1997) J. Biol. Chem. 272:7182.
- Ong, E. et al. (1998). Glycobiology 8:415.
- Korima, N. et al. (1996) J. Biol. Chem. 271:19457.
- Thompson, M. G. et al. (2011) J. Biol. Chem. 286:4525.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
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