UniOne® TR-FRET Human BRD4/CRBN PROTAC Binding Kit

This kit utilizes Homogeneous Time-Resolved Fluorescence(TR-FRET) technology to measure the Molecular Glue BET Degrader-1 and testcompound-mediated interaction between the DDB1/CRBN complex and BRD4. BRD4contains bromodomain 1 and 2. This method enables a simple and rapidhigh-throughput format for detecting small molecules capable of mediating theinteraction between the DDB1/CRBN complex and BRD4.

As shown, the interaction between DDB1/CRBN and BRD4 isdetected using a Eu-labeled Anti-Tag1 Antibody (TR-FRET donor) and anAcceptor-labeled Anti-Tag2 Antibody (TR-FRET acceptor). As the Molecular GlueBET Degrader-1 mediates the interaction between DDB1/CRBN and BRD4, the donorand acceptor antibodies are brought into proximity. Excitation of the donor antibodytriggers Fluorescence Resonance Energy Transfer (FRET) to the acceptorantibody, resulting in a specific emission signal at 665nm. This specificsignal is proportional to the degree of interaction mediated by BET Degrader-1between DDB1/CRBN and BRD4. This homogeneous assay is simple to perform andrequires no washing steps.

*Repeated freeze-thaw cycles should be avoided. Aliquot upon first thaw and store according to the specified conditions. Valid for one year.

1Reagent Preparation

Thaw all reagents to room temperature before use (equilibrate at room temperature for at least30min).384-well shallow plate reaction volume is20uL (reagent volumes for the reaction system are shown in the table). Calculate the required volume for the experiment before preparation and prepare accordingly; the following preparation is for reference only, using500 assays as an example.

1.1 Preparation of Buffer (1×)

2mL Detection buffer (10×), add18mLsterile water, mix well, and set aside.

Table2. Reagent Preparation and Volumes

1.2 Mix-1Preparation

Anti-Tag1 Eu dye antibodyandAnti-Tag2 Ac antibodyafter diluting separately,1:1mix well, and set aside.

1.3 Sample2-Fold Serial Dilution

UsingBET Degrader-1as an example, the diluent is1× Detection buffer. To minimize matrix effect interference, it is recommended to dilute with a solution matching the matrix of the test sample; adjust the test sample concentration according to actual conditions.

Table3. Serial Dilution of Samples

2. Sample Addition and Controls

2.1 Addition order for assay wells:4uL Tag1-DDB1/CRBN protein,4μL Tag2- BRD4 protein,2μL BET Degrader-1or test sample,10μLof mixedMix-1, add sequentially into the384-well shallow plate.

2.2 Negative control wells (Blank): BET Degrader-1or test compound is replaced with2μLofDMSOsolution at the same percentage.

2.3 NC:16uL 1× buffer plus4μLof dilutedAnti-Tag1 Eu dye antibody.

Table4. Sample Addition and Controls

*For negative controls and quality control, it is recommended to replace1×Detection bufferwith a solution matching the matrix of the test sample.

3.Detection

Detect using aTR-FRET-compatible microplate reader (excitation wavelength:320nm; emission wavelengths:620nmand665nm).

[Result Calculation]

1. Calculate signal value:665nmfluorescence signal divided by620nmfluorescence signal, then multiplied by10000, i.e.,

Signal Value= (665/620)*10000

2. Calculate the δ value based on the signal value:

δ Value= (Std-NC)/NC*100

3.CalculateCV(%)

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

[Data Example]

The following data cannot replace data obtained from actual experiments and is provided for illustration only. Results may vary depending on theTR-FRET-compatible instrument used.