Recombinant Human B4GalT1 (Y285L) Protein, CF SummaryLearn more about Fluorescent Glycan Labeling and Detection
Gly44-Ser398, with an N-terminal 6-His tag and a Y285L mutation
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CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|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.
- 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
- Dilute UDP-GalNAc to 1 mM in Assay Buffer.
- Dilute Coupling Phosphatase 1 to 20 ng/µL in Assay Buffer.
- Dilute GlcNAc to 200 mM in Assay Buffer.
- Prepare Reaction Mixture by combining equal volumes of 1 mM UDP-GalNAc, 20 ng/µL Coupling Phosphatase 1, and 200 mM GlcNAc.
- Dilute rhB4GalT1 to 1 µg/mL in Assay Buffer.
- 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.
- 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.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Load 20 µL of the 1 µg/mL rhB4GalT1 into the plate. Include a Substrate Blank containing 20 µL of Assay Buffer.
- Start the reaction by adding 30 µL of Reaction Mixture to the wells, excluding the standard curve and curve blank.
- Seal plate and incubate at 37 °C for 20 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells.
- Add 100 µL of deionized water to all wells. Mix briefly.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- 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
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).
- Guo, S. et al. (2001) Glycobiology 11:813.
- Yamaguchi, N. and Fukuda, M.N. (1995) J. Biol. Chem. 270:12170.
- Ramakrishnan, B. and Qasba, P.K. (2002) J. Biol. Chem. 277:20833.
- Do, K.Y. et al. (1997) Glycobiology 7:183.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
Citation for Recombinant Human B4GalT1 (Y285L) Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
Towards Mapping of the Human Brain N-Glycome with Standardized Graphitic Carbon Chromatography
Authors: J Helm, L Hirtler, F Altmann
Sample Types: Small Molecule
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Glycan Detection Reagents
Glycosyltransferase Activity Assays
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