Recombinant Human GAL3ST2 Protein, CF 5 UG

• Purity

  >90%, 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 sulfate from PAPS to alpha -Lactose. The specific activity is >800 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Chinese Hamster Ovary cell line, CHO-derived His32-Ala398 with an N-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  His

• Predicted Molecular Mass

  43 kDa

• SDS-PAGE

  55-65 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 25 mM MES, 15 mM MgCl₂, pH 5.5

• Coupling Phosphatase Buffer: 100 mM Tris, 15 mM MgCl₂, pH 8.0

• Recombinant Human Galactose‑3‑O‑sulfotransferase 2/GAL3ST2 (rhGAL3ST2) (Catalog # 7719-ST)

• Donor Substrate: 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) (Catalog # )

• Acceptor Substrate: alpha -Lactose (Sigma, Catalog # L2643), 0.3 M stock in deionized water

• Universal Sulfotransferase Activity Kit  (Catalog # )

• 96-well Clear Plate (Costar, Catalog # 92592)

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. 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.

2. 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.

3. Prepare a Substrate mixture of 0.8 mM PAPS and 10 mM alpha -Lactose in Assay Buffer.

4. Dilute rhGAL3ST2 to 4 µg/mL in Assay Buffer.

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

6. Load 25 µL of the 4 µg/mL rhGAL3ST2 into the plate. Include a Control containing 25 µL of Assay Buffer.

7. Add 25 µL of Substrate mixture to the wells, excluding the standard curve.

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

9. Dilute Coupling Phosphatase 3 (supplied in kit) to 10 µg/mL in Coupling Phosphatase Buffer.

10. Add 50 µL of 10 µg/mL Coupling Phosphatase 3 to each well, excluding the standard curve.  Add Coupling Phosphatase Buffer to the standard curve.

11. Cover the plate with a plate sealer and incubate at 37 °C for 10 minutes.

12. Add 30 µL of the Malachite Green Reagent A to all wells.  Mix briefly.

13. Add 50 µL of deionized water to all wells. Mix briefly.

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

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

16. Calculate specific activity:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.
     **Use the incubation time of the reaction between the Enzyme and Substrate mixture only, exclude additional incubation times.

Per Reaction:

• rhGAL3ST2:  0.1 µg

• Coupling Phosphatase 3: 0.5 µg

• PAPS: 20 nmol

• alpha -Lactose: 250 nmol

Background: Galactose-3-O-sulfotransferase 2/GAL3ST2

Galactose-3-O-sulfotransferase 2 (GAL3ST2) is a type II Golgi resident transmembrane protein. It transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the C-3 hydroxyl group of nonreducing beta -galactosyl residues present on various glycans, such as N-acetyllactosamine, lactose, lacto-N-tetraose (Lc4), lacto-N-neotetraose (nLc4), type 1 oligosaccharides (Gal beta 1-3GlcNAc-R), type 2 oligosaccharides (Gal beta 1-4GlcNAc-R), and core 1 O-glycans (Gal beta 1-3GalNAc alpha 1-Ser/Thr) (1). Given its broad substrate specificity, GAL3ST2 is capable of radioisotope labeling of non-reducing terminal galactose on glycoproteins or glycolipids with   ³⁵S. In addition, it can be used to generate galactin-4 and galactin-8 specific ligands (2, 3). The protein is widely expressed in various tissues (1). The enzymatic activity of the recombinant human GAL3ST2 is measured using a phosphatase-coupled assay (4).

• References:

1. Honke, K. et al. (2001) J. Biol. Chem. 276: 267.

2. Ideo, H. et al. (2002) Glycobiology 12:199.

3. Ideo, H. et al. (2011) J. Biol. Chem. 286:11346.

4. Prather, B. et al. (2012) Anal. Biochem. 423:86.

• Entrez Gene IDs:

  64090 (Human); 381334 (Mouse); 685402 (Rat)

• Alternate Names:

  GAL3ST2; Galactose-3-O-sulfotransferase 2; GP3ST