Recombinant Human Blood Group B Transferase/GTB Protein, CF
Recombinant Human Blood Group B Transferase/GTB Protein, CF Summary
Product Specifications
Arg63-Pro354, with an N-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
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.
|
Assay Procedure
- 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
- 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.
- Dilute UDP-galactose to 1.5 mM in Assay Buffer.
- Dilute Coupling Phosphatase I to 12 ng/μL in Assay Buffer.
- Dilute LNFP1 to 4.8 mM in Assay Buffer.
- Prepare substrate mixture by combining equal volumes of 1.5 mM UDP-galactose, 12 ng/μL Coupling Phosphatase I and 4.8 mM LNFP1.
- Dilute rhGTB to 1 µg/mL in Assay Buffer.
- 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.
- 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 25 µL of the 1 µg/mL rhGTB into the plate. Include a Control containing 25 µL of Assay Buffer.
- Add 25 µL of substrate mixture (step 4) to the wells, excluding the standard curve.
- Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- 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 Reaction:- rhGTB: 0.025 µg
- UDP-galactose: 250 µM
- Coupling Phosphatase I: 0.100 µg
- LNFP1: 800 µM
Reconstitution Calculator
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).
- Marcus, S.L. et al. (2003) J. Biol. Chem. 278:12403.
- Persson, M. et al. (2007) J. Biol. Chem. 282:9564.
- Yamamoto, F. et al. (1990) J. Biol. Chem. 265:19257.
- Yamamoto, F. et al. (1990) Nature 345:229.
- Patenaude, S.I. et al. (2002) Nat. Struct. Biol. 9:685.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
Citation for Recombinant Human Blood Group B Transferase/GTB 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
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In Depth Breadth Analyses of Human Blockade Responses to Norovirus and Response to Vaccination
Authors: J Haynes, V Perry, E Benson, A Meeks, G Watts, H Watkins, R Braun
Viruses, 2019-04-26;11(5):.
Species: Porcine
Sample Types: Protein
Applications: Bioassay
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