APEG1 Protein, Human (His)

Materials
Biotin
Pre-chilled sterile water
Wash buffer: 1X PBST
Blocking buffer: 1% BSA in 1X PBST
TMB substrate solution: Mix substrate solution A and B at a 1:1 ratio immediately before use
Stop solution: 2 M H₂SO₄
APEG1 Protein, Human (His) (HY-P76729)

Procedure
1. Coating: Dilute Human APEG1 to 2 μg/mL in 1X PBST, add 100 μL/well, and incubate overnight at 2-8°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, incubate at 25°C with shaking at 450 rpm for 4 hours.
4. Dilute the primary antibody to concentrations of 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 50, 100, and 250 μg/mL.
5. Add primary antibody: 100 μL/well. Incubate for 2 hours at 25°C, 450 rpm.
6. Wash plates: Wash plates with wash buffer, 260 μL/well. Wash five times, allowing each wash to stand for 1 minute, then blot dry.
7. Add secondary antibody: Dilute secondary antibody at a 1:2000 ratio. Add 100 μL per well and incubate at 25°C, 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 the OD value as the vertical axis and the Anti-APEG1 Antibody concentration as the horizontal axis, and calculate the ED₅₀ value.