Recombinant Human ST8SIA1 Protein, CF SummaryLearn more about Fluorescent Glycan Labeling and Detection
Tyr49-Ser356, 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 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, 5 mM MgCl2, pH 6.5
- Recombinant Human alpha -2,8‑Sialyltransferase 8A/ST8SIA1 (rhST8SIA1) (Catalog # 6716-GT)
- Donor Substrate: CMP-Neu5Ac (Sigma, Catalog # C8271), 10 mM stock in deionized water
- Acceptor Substrate: Fetuin (Sigma, Catalog # F3385), 50 mg/mL stock 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 nmoles per well.
- Dilute CMP-Neu5Ac to 2.5 mM in Assay Buffer.
- Dilute Coupling Phosphatase 2 to 25 µg/mL in Assay Buffer.
- Prepare reaction mixture by combining 50 µL of 2.5 mM CMP-Neu5Ac, 50 µL of 25 µg/mL Coupling Phosphatase 2, 62.5 µL of 50 mg/mL Fetuin, and 150 µL of Assay Buffer.
- Dilute rhST8SIA1 to 40 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 40 µg/mL rhST8SIA1 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:
- rhST8SIA1: 1.0 µg
- Coupling Phosphatase 2: 0.1 µg
- Fetuin: 250 µg
- CMP-Neu5Ac: 0.2 mM
Background: ST8 alpha-2,8-Sialyltransferase 8A/ST8SIA1
Gangliosides are acidic glycosphingolipids that contain one or more sialic acid residues and are particularly prevalent on neuronal cells (1). Ganglioside GD3 is involved in cell adhesion and the growth of cultured malignant cells (2). ST8SIA1 is a sialyltransferase that catalyzes the transfer of sialic acid from CMP-sialic acid to GM3 (NeuNAc alpha 2‑3Gal beta 1‑4Glc‑Cer) to produce GD3 (NeuNAc alpha 2‑8NeuNAc alpha 2‑3Gal beta 1‑4Glc‑Cer) and GT3 (NeuNAc alpha 2‑8NeuNAc alpha 2‑8NeuNAc alpha 2‑3Gal beta 1‑4Glc‑Cer) in a successive manner (3); therefore the enzyme has both GD3 and GT3 synthase activity (4). ST8SIA1 is mainly expressed in adult and fetal brain, and its expression is enhanced in melanoma cell lines (3, 4, 5). Like most known glycosyltransferases, ST8SIA1 is predicted as a type II transmembrane protein with a short N‑terminal cytoplasmic domain and a single-pass transmembrane domain followed by an enzymatic domain in the lumen of the Golgi apparatus. However, recently GD3 synthase activity was demonstrated at the surface of epithelial and melanoma cells, suggesting glycosphingolipid synthesis may occur at the cell membrane (6). Recombinant ST8SIA1 also showed activity on fetuin from fetal calf serum, when measured using a phosphatase-coupled method (7).
- Kolter, T. et al. (2002) J. Biol. Chem. 277:25859.
- Cheresh, D.A. et al. (1986) J. Cell. Biol. 102:688.
- Nakayama, J. et al. (1986) J. Biol. Chem. 271:3684.
- Nara, K. et al. (1994) Proc. Natl. Acad. Sci. USA. 91:7952.
- Haraguchi, M. et al. (1994) Proc. Natl. Acad. Sci. USA. 91:10455.
- Crespo, P.M. et al. (2010) J. Biol. Chem. 285:29179.
- Wu, Z.L. et al. (2010) Glycobiology doi: 10.1093/glycob/cwq187.
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