PCSK9 Protein, Human (HEK293, V474I, G670E, His)

Materials
PCSK9 Protein, Human (HEK293, V474I, G670E, His) （HY-P70545）
Biotin
Pre-cooled Sterile Water
Washing Buffer: 1×PBST
Blocking Buffer: PBST with 1% BSA
TMB Chromogenic Solution: Mix Chromogenic Solution A and Chromogenic Solution B at a ratio of 1:1 before use; prepare freshly as needed
Stop Solution: 2 M H₂SO₄

Procedure
Biotin Labeling
1. Dissolution of Biotin reagent: Remove Biotin from -10~-30°C, immediately dissolve 1 tube of Biotin with 180 μL of cold pure water. Gently pipette to mix, preparing 10 mM Biotin.
2. Calculate the volume of EZ-Link^TM Sulfo-NHS-LC-LC-Biotin, No-Weigh^TM Format to be added.
3. Incubate at room temperature in the dark for 45 min.
4. Quantification of labeled protein: Perform protein concentration quantification using BCA assay. The concentration of the labeled Biotinylated Human PCSK9 is 1670 μg/mL.

ELISA Detection
1. Coating: Dilute Recombinant Human LDLR protein to 2 μg/mL with 1×PBS, add 100 μL/well, and incubate at 4°C overnight.
2. Plate washing: Wash the plate with washing buffer, 260 μL/well, once, then pat dry.
3. Blocking: Use blocking buffer (1×PBST containing 1% BSA), 150 μL/well, block at 25°C with 450 rpm for 4 h.
4. Dilute Biotinylated Human PCSK9 to the following concentrations: 0, 0.25, 0.5, 2.5, 5, 25, 50, 250, 500, 2500, 5000, 25000, 50000 ng/mL.
5. Add Biotinylated Human PCSK9: 100 μL/well, incubate at 25°C with 450 rpm for 2 h.
6. Plate washing: Wash the plate with washing buffer, 260 μL per well, 5 times, with 1 min retention each time, then pat dry.
7. Add secondary antibody: Dilute the secondary antibody at a ratio of 1:2000, 100 μL/well, incubate at 25°C with 450 rpm for 1 h.
8. Plate washing: Wash the plate with washing buffer, 260 μL/well, 3 times, with 1 min retention each time, then pat dry.
9. Add TMB chromogenic solution: 100 μL/well; incubate at room temperature in the dark for 15 min.
10. Terminate the reaction: Add 2 M H₂SO₄, 100 μL/well.
11. Plate reading: Read at 450 nm.