CMP-Neu5Ac (Sigma, Catalog # C8271), 10 mM stock in deionized water
Fetuin (Sigma, Catalog # F3385), 50 mg/mL stock in deionized water
96-well Clear Plate (Costar, Catalog # 92592)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Prepare 1X Assay Buffer containing 10 mM MnCl2 by combining equal volumes of 10X Assay Buffer and 100 mM MnCl2 and diluting 5-fold with deionized water.
Dilute the 1 mM Phosphate Standard (provided in kit) by adding 40 μL of the 1 mM 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 a reaction mixture containing 0.5 mM CMP-Neu5Ac, 20 mg/mL Fetuin, and 4 μg/mL Coupling Phosphatase 2 in 1X Assay Buffer.
Dilute rmST6GALNAC2 to 2.4 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 2.4 µg/mL rmST6GALNAC2 into empty wells of the same plate containing the standard curve. Include a Control containing 25 µL of 1X 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 and incubate for 10 minutes at room temperature.
Add 100 µL of deionized water to all wells.
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) =
Adjusted 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.
rmST6GALNAC2: 0.060 μg
Coupling Phosphatase 2: 0.1 μg
CMP-Neu5Ac: 250 µM
Fetuin: 0.5 mg
Background: ST6 Sialyltransferase 2/ST6GALNAC2
Sialic acid molecules attached to glycoproteins or glycosphingolipids play important roles in various biological processes such as immune recognition, pathogen infection, and cell adhesion. Sialyltransferases are key enzymes that regulate the serum levels of sialic acid‑containing molecules. Alpha‑N‑acetylgalactosaminide alpha ‑2,6‑sialyltransferase 2, encoded by the ST6GALNAC2 gene, is a type II membrane protein localized in the Golgi network and catalyzes 2,6‑sialylation of the Tn antigen (GalNAc‑O‑Ser/Thr) (1). Sialyl‑Tn antigen is highly expressed in several human carcinomas and is associated with carcinoma aggressiveness and poor prognosis. Cells transfected with ST6GALNAC2 synthesized the sialyl‑6T structure [Gal beta 1‑3 (Neu5Ac alpha 2‑6)GalNAc‑O‑Ser/Thr] (2). In vitro analysis revealed that the enzyme can use both Gal beta 1‑3GalNAc and Neu5Ac alpha 2‑3Gal beta 1-3GalNAc as acceptor substrates (3). Recently, it has been reported that disregulation of this gene is involved in the pathogenesis of IgA nephropathy, a common kidney disease that occurs when antibody IgA accumulates in kidneys (4). The enzyme may also be used to synthesize a cancer vaccine (5, 6). At the protein level, mouse ST6GALNAC2 shows 75% amino acid sequence identity with the human homologue. The activity of this enzyme has been measured using a phosphatase‑coupled assay (7).
Kono, M., et al. (2000) Biochem. Biophys. Res. Commun. 272:94.
Marcos, N.T., et al. (2004) Cancer Res. 64:7050.
Samyn-Petit, B., et al. (2000) Biochim. Biophys. Acta 1474:201.
Li, G.S., et al. (2007) Hum. Mutat. 28:950.
Julien, S., et al. (2009) Br. J. Cancer 100:1746.
Sewell, R., et al. (2006) J. Biol. Chem. 281:3586.
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