TrkC Protein, Human (HEK293, His)

Materials
Biotin
Pre-chilled sterile water
Test protein: TrkC Protein, Human (HEK293, His) (HY-P73465)
Ligand protein: Neurotrophin-3 Protein, Human (HY-P70456)
Washing Buffer: 1XPBST
Blocking Solution: 1% BSA in PBST
TMB Substrate Solution: Mix Substrate Solution A and Substrate Solution B at a 1:1 ratio immediately before use
Stop Solution 2 M H₂SO₄

Procedure
1. Coating: Dilute Human NT-3 protein to 2 μg/mL with 1X PBS, add 100 μL/well, and incubate overnight at 4°C.
2. Washing: Wash the plate with 260 μL/well of wash buffer, wash once, and pat the plate dry.
3. Blocking: Use 1% BSA in 1X PBST (blocking solution), 150 μL/well, at 25°C, 450 rpm for 4 hours.
4. Dilute Human TrkC to concentrations of 0, 0.001, 0.01, 0.1, 0.5, 1, 2.5, 5, 10, 50, 100, and 5000 ng/mL.
5. Add receptor protein: 100 μL/well, incubate at 25°C, 450 rpm for 2 hours.
6. Washing: Wash the plate with 260 μL/well of wash buffer, wash 5 times, with a 1-minute dwell time each, then pat the plate dry.
7. Add secondary antibody: Dilute secondary antibody at a 1:2000 ratio, 100 μL/well; incubate at 25°C, 450 rpm for 1 hour.
8. Washing: Wash the plate with 260 μL/well of wash buffer, wash 3 times, with a 1-minute dwell time each, then pat the plate dry.
9. Add TMB substrate solution: 100 μL/well; incubate at room temperature in the dark for 15 minutes.
10. Stop the reaction: Add 2 M H₂SO₄, 100 μL/well.
11. Read the plate: Measure absorbance at 450 nm.