UniOne® TR-FRET Human BCL-XL/CRBN PROTAC Binding Kit

The assay kit employs homogeneous time-resolved fluorescence (TR-FRET) technology to measure the interaction between the DDB1/CRBN complex and BCL-XL mediated by the molecular glue XZ739 and test compounds. This method enables a simple, rapid, and high-throughput screening of small molecules capable of mediating the interaction between the DDB1/CRBN complex and BCL-XL.

As shown in the figure below, the interaction between DDB1/CRBN and BCL-XL is detected using a Eu-labeled anti-Tag1 antibody (TR-FRET donor) and an acceptor-labeled anti-Tag2 antibody (TR-FRET acceptor). The molecular glue XZ739 mediates the interaction between DDB1/CRBN and BCL-XL, bringing the donor and acceptor antibodies into proximity. Excitation of the donor antibody triggers fluorescence resonance energy transfer (FRET) to the acceptor antibody, resulting in a specific emission signal at 665 nm. This signal is proportional to the extent of interaction mediated by XZ739 with DDB1/CRBN and BCL-XL. The homogeneous assay is simple to perform and requires no washing steps.

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Note: Aliquot all components immediately after first thawing and store at the recommended temperature. Avoid storage after dilution and repeated freeze-thaw cycles.

1. Reagent Preparation

1.1 Before use, thaw all reagents at room temperature (equilibrate at room temperature for at least 30 min). The reaction volume for a 384-well shallow well plate is 20 μL (reagent volumes for the reaction system are shown in the table). Calculate the required volume before preparation and prepare as needed; the following preparation is for reference only, using 500 tests as an example.

Table 1. Reagent Preparation

1.2 Gradient Dilution of Test Samples

Using XZ739 as an example, the diluent is 1× Detection buffer. To minimize matrix effect interference, it is recommended to dilute with a solution matching the test sample matrix; adjust the test samples according to actual concentrations.

Table 2. XZ739 Gradient Dilution (Adjust According to Actual Conditions)

2. Sample Addition and Controls

2.1 Test Samples: Add 4 μL Tag1-CRBN protein working solution, 4 μL Tag2-BCL-XL protein working solution, 2 μL gradient-diluted test samples, and 10 μL mixed Antibody Mix sequentially into the 384-well shallow well plate.

2.2 Positive Control Standard Curve: 4 μL Tag1-CRBN protein working solution, 4 μL Tag2-BCL-XL protein working solution, 2 μL gradient-diluted XZ739, and 10 μL Antibody Mix.

2.3 Blank Control Wells: Replace test samples with 2 μL 1× Detection buffer;

2.4 NC (Negative Control): Add 10 μL 1× Detection buffer and 10 μL Antibody Mix.

After adding all samples, centrifuge, seal with a plate sealing film, and incubate at room temperature for 2 hours.

3. Detection

Detect using a microplate reader compatible with TR-FRET. Excitation wavelength is 320/340 nm, and emission wavelengths detected are 620 nm and 665 nm.

[Result Calculation]

1) Calculate signal value (Ratio): Divide the fluorescence signal at 665 nm by the signal at 620 nm, then multiply by 10000.

Ratio = (665/620) ×10000

2) Calculate 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 experimental results and is provided only as an example; results may vary depending on the plate reader used.

3 /3Note: Recommended microplates (384-well, white, shallow well);