Recombinant Human ST8SIA4 Protein, CF
Recombinant Human ST8SIA4 Protein, CF Summary
Gly40-Gln359, 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, NaCl and Brij-35.|
|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 Buffer: 100 mM MES, 10 mM MnCl2 (supplied in kit), pH 7.0
- Recombinant Human alpha -2,8-Sialyltransferase 4/ST8SIA4 (rhST8SIA4) (Catalog # 7027-GT)
- Donor Substrate: CMP-Sialic Acid (Sigma, Catalog # C8271), 10 mM stock in deionized water
- Acceptor Substrate: Recombinant Human NCAM-1/CD56 120 isoform (rhNCAM-1) (Catalog # 2408-NC)
- Sialyltransferase Activity Kit (R&D Systems, Catalog # EA002)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- 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.
- Prepare 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 nmol per well.
- Reconstitute lyophilized rhNCAM-1/CD56 to 500 μg/mL in deionized water, 80 μg is needed for this protocol (do not reconstitute in PBS).
- Dilute CMP-Sialic Acid to 2.78 mM in Assay Buffer.
- Dilute Coupling Phosphatase 2 to 22.2 µg/mL in Assay Buffer.
- Prepare reaction mixture by combining 45 µL of 2.78 mM CMP-Sialic Acid, 45 µL of 22.2 µg/mL Coupling Phosphatase 2, and 160 µL of 500 μg/mL rhNCAM-1/CD56. (Volume is sufficient to test 10 wells.)
- Dilute rhST8SIA4 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 rhST8SIA4 into the plate. Include a Control containing 25 µL of Assay Buffer.
- Start the reaction by adding 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:
- rhST8SIA4: 0.5 μg
- Coupling Phosphatase 2: 0.1 μg
- rhNCAM-1/CD56: 8 μg
- CMP-Sialic Acid: 0.25 mM
Background: ST8 alpha-2,8-Sialyltransferase 4/ST8SIA4
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). The temporal regulation of PSA is dependent on the expression of two polysialyltransferases, ST8SIA4 and ST8SIA2 (4, 5). While ST8SIA2 shows strict preference for NCAM (6), ST8SIA4 is also active on other glycoproteins, such as fetuin, in vitro (7). Unlike ST8SIA2, ST8SIA4 can catalyze both the initiation and polymerization of PSA. A combination of ST8SIA2 and ST8SIA4 cooperatively polysialylate NCAM, resulting in PSA with longer chains than when the enzymes are used individually (8). Disruption of ST8SIA4 in mice leads to impaired long-term potentiation and long-term depression of synapses of the adult hippocampus, similar to the phenotypes observed in NCAM‑deficient mice (9). Like most glycosyltransferases, ST8SIA4 may be a Golgi‑resident type II membrane protein. The activity of this enzyme has been measured with a phosphatase-coupled method (10).
- 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.
- Nakayama, J. and Fukuda, M. (1996) J. Biol. Chem. 271:1829.
- Angata, K. and Fukuda, M. (2002) J. Biol. Chem. 277:36808.
- Eckhardt, M. et al. (2000) J. Neurosci. 20:5234.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
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Glycosyltransferase Activity Assays
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