Recombinant Human B4GalT1 (Y285L) Protein, CF

R&D Systems | Catalog # 7040-GT

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Key Product Details

  • R&D Systems NS0-derived Recombinant Human B4GalT1 (Y285L) Protein (7040-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

NS0

Accession Number

P15291

Applications

Enzyme Activity
View all beta-1,4-Galactosyltransferase 1/B4GalT1 Proteins and Enzymes »

Product Specifications

Source

Mouse myeloma cell line, NS0-derived human beta-1,4-Galactosyltransferase 1/B4GalT1 protein
Gly44-Ser398, with an N-terminal 6-His tag and a Y285L mutation

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

40 kDa

SDS-PAGE

45-60 kDa, reducing conditions

Activity

Measured by its ability to transfer N-Acetyl-D-galactosamine from UDP-GalNAc to N-Acetyl-D-glucosamine.
The specific activity is >2,000 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

7040-GT
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and TCEP.
Shipping The product is shipped with polar packs. 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, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: beta-1,4-Galactosyltransferase 1/B4GalT1

beta ‑1,4‑Galactosyltransferase 1, also known as B4GalT1, is one of seven beta ‑1,4 galactosyltransferases that transfer galactose to acceptor sugars including GlcNAc, Glc and Xyl (1, 2). A single point mutation, Y285L, in beta 4GalT1 causes a functional switch in activity from Gal‑transferase activity to GalNAc‑transferase activity (3).The recombinant human B4GalT1(Y285L) can be used to synthesize lacdiNAc-terminated glycoconjugates, terminal glycan modifications found on many glycoproteins, including activation receptors of natural killer cells, NKR-P1 and CD69 (4). The enzymatic activity of the recombinant human B4GalT1 (Y285L) was assayed using a phosphatase-coupled method (5).

References

  1. Guo, S. et al. (2001) Glycobiology 11:813.
  2. Yamaguchi, N. and Fukuda, M.N. (1995) J. Biol. Chem. 270:12170.
  3. Ramakrishnan, B. and Qasba, P.K. (2002) J. Biol. Chem. 277:20833.
  4. Do, K.Y. et al. (1997) Glycobiology 7:183.
  5. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Long Name

UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, Polypeptide 1

Alternate Names

GGTB2

Entrez Gene IDs

2683 (Human); 14595 (Mouse); 24390 (Rat)

Gene Symbol

B4GALT1

UniProt

P15291

Additional beta-1,4-Galactosyltransferase 1/B4GalT1 Products

Product Documents for Recombinant Human B4GalT1 (Y285L) Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

Download SDS

Product Specific Notices for Recombinant Human B4GalT1 (Y285L) Protein, CF

For research use only

Related Research Areas

Glycobiology-related Enzymes

Citations for Recombinant Human B4GalT1 (Y285L) Protein, CF

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Protocols

View specific protocols for Recombinant Human B4GalT1 (Y285L) Protein, CF (7040-GT):

Materials
  • Assay Buffer: 50 mM HEPES, 10 mM MnCl2 (supplied in kit), pH 7.5
  • Recombinant Human beta ‑1,4‑Galactosyltransferase 1/B4GalT1 (Y285L) (rhB4GalT1) (Catalog # 7040-GT)
  • Donor Substrate: Uridine 5’-diphospho-N-acetylgalactosamine (UDP-GalNAc) (Sigma, Catalog # U5252), 10 mM stock in deionized water
  • Acceptor Substrate: N-Acetyl-alpha -D-glucosamine (GlcNAc) (Calbiochem, Catalog # 1079), 1 M stock in deionized water
  • Glycosyltransferase Activity Kit (Catalog # EA001)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute UDP-GalNAc to 1 mM in Assay Buffer.
  2. Dilute Coupling Phosphatase 1 to 20 ng/µL in Assay Buffer.
  3. Dilute GlcNAc to 200 mM in Assay Buffer.
  4. Prepare Reaction Mixture by combining equal volumes of 1 mM UDP-GalNAc, 20 ng/µL Coupling Phosphatase 1, and 200 mM GlcNAc.
  5. Dilute rhB4GalT1 to 1 µg/mL in Assay Buffer.
  6. 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. This is the first point of the standard curve.
  7. 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.
  8. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  9. Load 20 µL of the 1 µg/mL rhB4GalT1 into the plate. Include a Substrate Blank containing 20 µL of Assay Buffer.
  10. Start the reaction by adding 30 µL of Reaction Mixture to the wells, excluding the standard curve and curve blank.
  11. Seal plate and incubate at 37 °C for 20 minutes.
  12. Add 30 µL of the Malachite Green Reagent A to all wells.
  13. Add 100 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:

     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 Well:

  • rhB4GalT1:  0.020 µg
  • Coupling Phosphatase 1: 0.2 µg
  • UDP-GalNAc: 10 nmol
  • GlcNAc: 2000 nmol

FAQs

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