• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to transfer Neu5Ac from CMP-Neu5Ac to fetuin of fetal calf serum. The specific activity is >700 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Mouse myeloma cell line, NS0-derived Ser28-Arg373, with an N-terminal human CD33 signal sequence and a C-terminal 6?His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ser28

• Predicted Molecular Mass

  40 kDa

• SDS-PAGE

  40-50 kDa, reducing conditions

Assay Procedure

Materials

• Sialyltransferase Activity Kit (Catalog # )

• 10X Assay Buffer (supplied in kit): 250 mM Tris, pH 7.5

• Recombinant Mouse Sialyltransferase 2/ST6GALNAC2  (rmST6GALNAC2) (Catalog # 6468-GT)

• 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

1. 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.

2. 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.

3. 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.

4. 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.

5. Dilute rmST6GALNAC2 to 2.4 μg/mL in 1X Assay Buffer.

6. Load 50 μL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.

7. 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.

8. Add 25 μL of reaction mixture to the wells, excluding the standard curve.

9. Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.

10. Add 30 μL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.

11. Add 100 μL of deionized water to all wells.

12. Add 30 μL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.

13. Read plate at 620 nm (absorbance) in endpoint mode.

14. Calculate specific activity:

     *Derived from the phosphate standard curve using linear fitting and adjusted for Control.

Per Reaction:

• 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).

• References:

1. Kono, M., et al. (2000) Biochem. Biophys. Res. Commun. 272:94.

2. Marcos, N.T., et al. (2004) Cancer Res. 64:7050.

3. Samyn-Petit, B., et al. (2000) Biochim. Biophys. Acta 1474:201.

4. Li, G.S., et al. (2007) Hum. Mutat. 28:950.

5. Julien, S., et al. (2009) Br. J. Cancer 100:1746.

6. Sewell, R., et al. (2006) J. Biol. Chem. 281:3586.

7. Wu, Z.L., et al. (2011) Glycobiology 21:727.

• Long Name:

  ST6 alpha-N-acetylGalactosaminide alpha-2,6-sialyltransferase 2

• Entrez Gene IDs:

  10610 (Human)

• Alternate Names:

  (alpha-N-acetylneuraminyl-2,3-beta-galactosyl-1,3)-N-acetyl galactosaminide B; alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase 2; EC 2.4.99.-; FLJ45660; GalNAc alpha-2,6-sialyltransferase II; SAITL1; sialyltransferase 7((alpha-N-acetylneuraminyl-2,3-beta-galactosyl-1,3)-N-acetyl galactosaminide B; sialyltransferase 7((alpha-N-acetylneuraminyl-2,3-beta-galactosyl-1,3)-N-acetyl galactosaminidealpha-2,6-sialyltransferase) B; sialyltransferase 7((alpha-N-acetylneuraminyl-2,3-beta-galactosyl-1,3)-N-acetyl galactosaminidealpha-2,6-sialyltransferase); Sialyltransferase 7B; sialyltransferase-like 1; SIAT7; SIAT7B; SIAT7-B; SIATL1; ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminidealpha-2,6-sialyltransferase 2; ST6GalNAc II; ST6GALNAC2; ST6GalNAcII; ST6GalNAII; STHM; STHM(alpha-N-acetylneuraminyl-2,3-beta-galactosyl-1,3)-N-acetyl galactosaminidealpha-2,6-sialyltransferase