Alkaline Phosphatase/ALPP Protein, Human (HEK293, C-His)

Materials
Alkaline Phosphatase/ALPP Protein, Human (HEK293, C-His) (HY-P7487A)
Wash buffer: 1X PBST
Blocking buffer: 1% BSA in 1X PBS-T
TMB Substrate Solution: Mix Substrate Solution A and B at a 1:1 ratio immediately before use
Stop Solution: 2 M H₂SO₄

Procedure
1. Coating: Dilute the test protein to 1 μg/mL in 1X PBS, add 100 μL/well, and incubate overnight at 4°C. 2. Washing: Wash the plate with wash buffer (260 μL/well) , once, and pat dry.
3. Blocking: Add 150 μL/well of blocking buffer (1% BSA in 1X PBST) , incubate at 25°C with shaking at 450 rpm for 4 hours.
4. Dilute Primary Antibody: Dilute the antibody to concentrations of 0, 0.01, 0.1, 1, 5, 10, 50, 100, 500, 1000, 5000, 10000, 50000 ng/mL.
5. Add Primary Antibody: Add 100 μL/well, incubate at 25°C with shaking at 450 rpm for 2 hours. 6. Washing: Wash the plate with wash buffer (260 μL/well) , five times, with each wash lasting 1 minute, then pat dry.
7. Add Secondary Antibody: Add 100 μL/well, incubate at 25°C with shaking at 450 rpm for 1 hour.
8. Washing: Wash the plate with wash buffer (260 μL/well) , three times, with each wash lasting 1 minute, then pat dry.
9. Add TMB Substrate Solution: Add 100 μL/well, incubate at room temperature in the dark for 15 minutes.
10. Stop Reaction: Add 100 μL of 2 M H₂SO₄ per well.
11. Read Plate: Measure absorbance at 450 nm.
12. Using GraphPad Prism software, plot a curve with OD values on the vertical axis and primary antibody concentration on the horizontal axis to calculate the ED₅₀ value.