Recombinant Human Blood Group B Transferase/GTB Protein, CF

R&D Systems | Catalog # 6824-GT

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Key Product Details

  • R&D Systems CHO-derived Recombinant Human Blood Group B Transferase/GTB Protein (6824-GT)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

CHO

Accession Number

AAD26574

Applications

Enzyme Activity
View all Blood Group B Transferase/GTB Proteins and Enzymes »

Product Specifications

Source

Chinese Hamster Ovary cell line, CHO-derived human Blood Group B Transferase/GTB protein
Arg63-Pro354, with an N-terminal 6-His tag

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

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

N-terminal Sequence Analysis

His

Predicted Molecular Mass

34.8 kDa

SDS-PAGE

35-40 kDa, reducing conditions

Activity

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.

Formulation, Preparation, and Storage

6824-GT
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.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Background: Blood Group B Transferase/GTB

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).

References

  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.

Long Name

Fucosylglycoprotein 3-alpha-galactosyltransferase

Alternate Names

A3GALT1, ABO

Entrez Gene IDs

28 (Human); 80908 (Mouse); 65270 (Rat)

Gene Symbol

ABO

UniProt

AAD26574

Additional Blood Group B Transferase/GTB Products

Product Documents for Recombinant Human Blood Group B Transferase/GTB Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human Blood Group B Transferase/GTB Protein, CF

For research use only

Related Research Areas

Glycobiology-related Enzymes

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

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Protocols

View specific protocols for Recombinant Human Blood Group B Transferase/GTB Protein, CF (6824-GT):

Materials
  • HEPES free acid (Recommended: Amresco, Catalog # 0511)
  • 100 mM MnCl2 (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

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