Recombinant Human Blood Group B Transferase/GTB Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Human Blood Group B Transferase/GTB Protein, CF Summary

Details of Functionality
Measured by its ability to transfer galactose from UDP-galactose to lacto-N-fucopentaose. The specific activity is >1,250 pmol/min/μg, as measured under the described conditions.
Source
Chinese Hamster Ovary cell line, CHO-derived human Blood Group B Transferase/GTB protein
Arg63-Pro354, with an N-terminal 6-His tag
Accession #
N-terminal Sequence
His
Protein/Peptide Type
Recombinant Enzymes
Gene
ABO
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
34.8 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
35-40 kDa, reducing conditions
Publications
Read Publication using
6824-GT in the following applications:

Packaging, Storage & Formulations

Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
Assay Procedure
  • HEPES free acid (Recommended: Amresco, Catalog # 0511)
  • 100 mM MnCl(Supplied in kit)
  • Deionized water
  • Recombinant Human Blood Group B Transferase/GTB (rhGTB) (Catalog # 6824-GT)
  • Donor Substrate: UDP-galactose (Sigma, Catalog # U4500), 10 mM stock in deionized water
  • Acceptor Substrate: Lacto-N-fucopentaose I (LNFP1) (V-Labs, Catalog # L502), 20 mM stock in deionized water
  • Glycosyltransferase Assay Kit (Catalog # EA001)
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare Assay Buffer by combining a Hepes stock, pH titrated with NaOH/HCl, with MnCl2 stock to yield: 50 mM Hepes, 5 mM MnCl2, pH 6.5. Note: fresh preparation required as Mn2+ will oxidize and precipitate with time.
  2. Dilute UDP-galactose to 1.5 mM in Assay Buffer.
  3. Dilute Coupling Phosphatase I to 12 ng/μL in Assay Buffer.
  4. Dilute LNFP1 to 4.8 mM in Assay Buffer.
  5. Prepare substrate mixture by combining equal volumes of 1.5 mM UDP-galactose, 12 ng/μL Coupling Phosphatase I and 4.8 mM LNFP1.
  6. Dilute rhGTB to 1 µg/mL in Assay Buffer.
  7. Dilute Phosphate Standard 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.
  8. Continue 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 5.0 nmol per well.
  9. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  10. Load 25 µL of the 1 µg/mL rhGTB into the plate. Include a Control containing 25 µL of Assay Buffer.
  11. Add 25 µL of substrate mixture (step 4) to the wells, excluding the standard curve.
  12. Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.
  13. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  14. Add 100 µL of deionized water to all wells. Mix briefly.
  15. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  16. Read plate at 620 nm (absorbance) in endpoint mode.
  17. 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:
  • rhGTB: 0.025 µg
  • UDP-galactose: 250 µM
  • Coupling Phosphatase I: 0.100 µg
  • LNFP1: 800 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Blood Group B Transferase/GTB Protein, CF

  • A transferase
  • A3GALNT
  • A3GALT1
  • ABO blood group (transferase A, alpha 1-3-N-acetylgalactosaminyltransferase;transferase B, alpha 1-3-galactosyltransferase)
  • ABO glycosyltransferase
  • ABO
  • B transferase
  • B(A) alpha-1,3-galactosyltransferase
  • EC 2.4.1.37
  • EC 2.4.1.40
  • Fucosylglycoprotein 3-alpha-galactosyltransferase
  • Fucosylglycoprotein alpha-N-acetylgalactosaminyltransferase
  • Glycoprotein-fucosylgalactoside alpha-galactosyltransferase
  • Glycoprotein-fucosylgalactoside alpha-N-acetylgalactosaminyltransferase
  • GTB
  • Histo-blood group A transferase
  • histo-blood group A2 transferase
  • histo-blood group ABO system transferase
  • Histo-blood group B transferase
  • NAGAT

Background

The ABO blood group type is characterized by the different carbohydrate antigens displayed on the cell surface of human erythrocytes. Blood group A exhibits the A antigen, GalNAc alpha 1-3(Fuc alpha 1-2)Gal. Blood group B exhibits the B antigen, Gal alpha 1-3(Fuc alpha 1-2)Gal. Blood group O exhibits the H antigen, Fuc alpha 1-2Gal. The H antigen is converted to either the A antigen via GTA, an alpha 1-3 N‑acetylgalactosaminyltransferase, or to the B antigen via GTB, an alpha 1-3 galactosyltransferase. GTA and GTB are encoded by different alleles of the ABO gene and differ at only four amino acids (R176G, G235S, L266M, and G268A). People with type O blood do not have a functional transferase expressed due to a frameshift mutation in the ABO gene. Both GTA and GTB are type II Golgi-resident transmembrane proteins but can also be found in secreted forms in body fluids (1, 2, 3, 4, 5). The activity of the recombinant human GTB was determined using a phosphatase-coupled glycosyltransferase assay (6).
  1. Marcus, S.L. et al. (2003) J. Biol. Chem. 278:12403.
  2. Persson, M. et al. (2007) J. Biol. Chem. 282:9564.
  3. Yamamoto, F. et al. (1990) J. Biol. Chem. 265:19257.
  4. Yamamoto, F. et al. (1990) Nature 345:229.
  5. Patenaude, S.I. et al. (2002) Nat. Struct. Biol. 9:685.
  6. Wu, Z.L. et al. (2011) Glycobiology 21:727.

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Publications for Blood Group B Transferase/GTB/ABO (6824-GT)(1)

We have publications tested in 1 confirmed species: Porcine.

We have publications tested in 1 application: Bioassay.


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Bioinformatics

Gene Symbol ABO
Uniprot