Recombinant Human Blood Group B Transferase/GTB Protein, CF

• 6 months from date of receipt, -70 °C as supplied.
• 3 months, -70 °C under sterile conditions after opening.

• 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

1. 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.
2. Dilute UDP-galactose to 1.5 mM in Assay Buffer.
3. Dilute Coupling Phosphatase I to 12 ng/μL in Assay Buffer.
4. Dilute LNFP1 to 4.8 mM in Assay Buffer.
5. Prepare substrate mixture by combining equal volumes of 1.5 mM UDP-galactose, 12 ng/μL Coupling Phosphatase I and 4.8 mM LNFP1.
6. Dilute rhGTB to 1 µg/mL in Assay Buffer.
7. 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.
8. 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.
9. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
10. Load 25 µL of the 1 µg/mL rhGTB into the plate. Include a Control containing 25 µL of Assay Buffer.
11. Add 25 µL of substrate mixture (step 4) to the wells, excluding the standard curve.
12. Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.
13. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
14. Add 100 µL of deionized water to all wells. Mix briefly.
15. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
16. Read plate at 620 nm (absorbance) in endpoint mode.
17. Calculate specific activity:

     *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