UniOne® TR-FRET Human PCSK9-LDLR Assay Kit

The kit employs homogeneous time-resolved fluorescence technology (TR-FRET) for measuring the interaction between Human PCSK9 and Human LDLR. This method enables simple, rapid, and high-throughput screening of inhibitors and antibody blockers.

As shown in the figure, the interaction between PCSK9 and LDLR is detected using an Eu-labeled anti-Tag1 antibody (TR-FRET donor) and an Ac-labeled Tag2 antibody (TR-FRET acceptor). The binding of PCSK9 to LDLR brings the donor and acceptor antibodies into proximity, allowing the excitation of the donor antibody to trigger fluorescence resonance energy transfer (FRET) to the acceptor antibody, resulting in a specific emission signal at 665 nm. Screened compounds, such as Pep 2-8, block the binding of PCSK9 to LDLR, preventing FRET signal generation. The stronger the blocking effect of the screened compound on PCSK9 and LDLR, the lower the signal. The signal is proportional to the degree of interaction between PCSK9 and LDLR. No washing steps are required.

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1. Reagent Preparation

1.1 Thaw all reagents at room temperature before use (allow at least 30 minutes for equilibration). The reaction system for the 384-well plate is 20μL (reagent volumes are shown in the table below). Calculate the required volume before preparation and prepare accordingly. The following preparation is for reference only, using 500 reactions as an example.

Table 1. Reagent Preparation

1.2 Gradient Dilution of Test Samples

Taking Pep 2-8 as an example, the diluent is 1× Detection buffer. To reduce the interference of matrix effects, it is recommended to use a solution with the same matrix as the test sample for dilution. The dilution of the test sample should be adjusted according to the actual concentration.

Table 2. Pep 2-8 Preparation Table (Adjust according to actual conditions)

2. Sample Loading and Controls

2.1 Test samples: Add 2μL gradient-diluted test samples, 4μL Tag2-PCSK9 protein, 4μL Tag1-LDLR protein working solution, and 10μL Antibody Mix sequentially into a 384-well shallow plate.

2.2 Blank control wells (Blank): Replace test samples with 2μL 1× Detection buffer.

2.3 Eu control wells (NC): Add 10μL 1× Detection buffer and 10μL Antibody Mix.

After all samples are loaded, centrifuge, cover with sealing film, and incubate at room temperature for 2 hours.

3、Detection

Perform detection on a TR-FRET-compatible microplate reader. Excitation wavelength is 320/340 nm, with emission wavelengths detected at 620 nm and 665 nm.

【Result Calculation】

1) Calculate the signal value (Ratio): Divide the 665 nm fluorescence signal by the 620 nm fluorescence signal and multiply by 10,000.

Ratio = (665/620) ×10000

2) Calculate the Net signal based on the signal value:

Net signal = (Std-NC)/NC×100

3) Calculate CV (%):

CV (%) = Standard Deviation/Mean Ratio × 100%

【Data Example】

The following data cannot replace the data obtained in experiments and is only provided as an example. Results may vary depending on the plate reader instrument. 

Note: Recommended microplate (384-well plate, white, shallow well)