GALNT7 Protein, Human (HEK293, His)

Materials
Assay buffer: 25 mM Tris, 5 mM MnCl₂, 2.5 mM CaCl₂, pH 7.5
GALNT7 Protein, Human (HEK293, His) (HY-P70867)
Substrates: UDP-GalNAc disodium (HY-114365), MUC5AC-3/13 Peptide
Glycosyltransferase Activity Kit

Procedure
1. Add 40 μL of 1 mM phosphate standard to 360 μL of assay buffer, and dilute the 1 mM phosphate standard provided in the glycosyltransferase kit to obtain a 100 μM stock solution.
2. Prepare a standard curve by performing six consecutive 1:2 dilutions of the 100 μM phosphate stock (diluted with assay buffer). The standard curve ranges from 0.078 to 5 nmol per well.
3. Prepare a reaction mixture consisting of 1 mM UDP-GalNAc, 0.4 mM MUC5AC-3/13 Peptide, and 8 ng/μL coupled phosphatase I in the assay buffer.
4. Dilute GALNT7 to 4 ng/μL with assay buffer.
5. Add 50 μL of different concentration dilutions of the standard curve to a 96-well plate, and add 50 μL of assay buffer to the blank group.
6. Add 25 μL of 4 ng/μL GALNT7 to the 96-well plate; add 25 μL of detection buffer to the blank group.
7. Add 25 μL of the reaction mixture to the wells, excluding the standard curve wells and curve blank wells.
8. Incubate at 37 ℃ for 20 minutes.
9. Add 30 μL of Malachite Green Reagent A to all wells and mix.
10. Add 100 μL of deionized water to all wells and mix.
11. Add 30 μL of Malachite Green Reagent B to all wells, mix, and incubate at room temperature for 20 minutes.
12. Read the plate at 620 nm (absorbance) in endpoint mode.
13. Calculate the specific activity: