B4GALT1 Protein, Human (HEK293, Y285L, His)

Materials
Assay buffer: 50 mM HEPES, 10 mM MnCl₂, pH 7.5
B4GALT1 Protein, Human (HEK293, Y285L, His) (HY-P7628)
Substrates: UDP-GalNAc (HY-112174), N-Acetyl-alpha-D-glucosamine (GlcNAc, HY-A0132)
Glycosyltransferase Activity Kit

Procedure
1. Dilute UDP-GalNAc to 1 mM with assay buffer.
2. Dilute Coupled Phosphatase 1 to 20 μg/mL with assay buffer.
3. Dilute GlcNAc to 200 mM with assay buffer.
4. Prepare the reaction mixture by mixing equal volumes of 1 mM UDP-GalNAc, 20 μg/mL Coupled Phosphatase 1, and 200 mM GlcNAc.
5. Dilute Human B4GalT1 to 1 μg/mL with assay buffer.
6. Add 40 μL of 1 mM phosphate standard to 360 μL of assay buffer to dilute the 1 mM phosphate standard provided in the Glycosyltransferase Activity Kit to obtain a 100 μM stock solution.
7. Prepare the standard curve by performing six 1:2 serial dilutions of the 100 μM phosphate stock solution in assay buffer. The standard curve range is 0.078 to 5 nmol/well.
8. Add 50 μL of different concentrations of the standard curve dilutions to a 96-well plate; add 50 μL of assay buffer to the blank group.
9. Add 20 μL of 1 μg/mL Human B4GalT1 to the 96-well plate; add 20 μL of assay buffer to the blank group.
10. Add 30 μL of the reaction mixture to the wells, excluding the standard curve wells and blank wells.
11. Incubate at 37 °C for 15 minutes.
12. Add 30 μL of Malachite Green Reagent A to all wells and mix.
13. Add 100 μL of deionized water to all wells and mix.
14. Add 30 μL of Malachite Green Reagent B to all wells, mix, and incubate at room temperature for 20 minutes.
15. Read the plate at 620 nm (absorbance) in endpoint mode.
16. Calculate Specific Activity: