>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
<1.0 EU per 1 μg of the protein by the LAL method.
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.
35-40 kDa, reducing conditions
Read Publication using 6824-GT in the following applications:
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.
rhGTB: 0.025 µg
UDP-galactose: 250 µM
Coupling Phosphatase I: 0.100 µg
LNFP1: 800 µM
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
ABO blood group (transferase A, alpha 1-3-N-acetylgalactosaminyltransferase;transferase B, alpha 1-3-galactosyltransferase)
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.
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