>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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PRODUCT AVAILABILITY: Update Regarding the Evolving COVID-19 Situation
Bio-Techne appreciates the critical role that you and our products and services play in research efforts to further scientific innovation and discovery. We are continually assessing our manufacturing and supplier capabilities during the COVID-19 situation and are implementing precautionary measures to ensure uninterrupted supply of products and services. Currently, and as we abide by local shelter in place orders across the world, we are fully operational and do not anticipate any material supply disruptions across our Bio-Techne brands and product lines. As the situation evolves, our goal is to utilize preventive measures to reduce the threat that COVID-19 poses to our ability to meet the needs of our customers globally.