UniOne® TR-FRET Human RAK4/CRBN PROTAC Binding KIT

This kit utilizes Time-Resolved Fluorescence ResonanceEnergy Transfer (TR-FRET) technology to measure the interaction between IRAK4and CRBN mediated by the molecular glue PROTAC IRAK4 DEGRADER-1 and testcompounds. This method provides a simple, rapid, and high-throughput format fordetecting small molecules capable of mediating IRAK4-CRBN interactions.

As shown, the interaction between IRAK4 and CRBN isdetected using an anti-Tag1 antibody labeled with Eu (TR-FRET donor) and ananti-Tag2 antibody labeled with Ac (TR-FRET acceptor). Since the molecular gluePROTAC IRAK4 DEGRADER-1 mediates the interaction between IRAK4 and CRBN, thedonor and acceptor antibodies are brought into proximity. Excitation of thedonor antibody triggers fluorescence resonance energy transfer (FRET) to theacceptor antibody, resulting in specific emission of a 665 nm signal. Thisspecific signal is proportional to the degree of interaction between PROTACIRAK4 DEGRADER-1, IRAK4, and CRBN. This homogeneous assay is simple to performand requires no wash steps.

*Avoid repeated freeze-thaw cycles of reagents. Recommend aliquoting after first thaw and storing according to conditions specified for a shelf life of one year.

1. Reagent Preparation

Thaw all reagents at room temperature (equilibrate at room temperature for at least 30 min) before use. The reaction system for a 384-well shallow plate is 20 μL (reagent volumes per well shown in table). Calculate the required volume based on your experimental needs and prepare accordingly. The following preparation is for reference only, using the 500-test kit as an example.

1.1 Buffer (1×):
Add 18 mL sterile water to 2 mL Detection buffer (10×) and mix well.

Table 2. Reagent Preparation and Volumes

1.2 Dilution of Test Samples

Using PROTAC IRAK4 DEGRADER-1 as an example, diluent is 1× Detection buffer. To minimize matrix interference, it is recommended to dilute with the same matrix as the test sample; adjust based on actual sample concentration.

Table 3. Sample Dilution Series

2. Sample and Control Setup

2.1 Pipetting order for assay wells: Add 4 μL Tag1-IRAK4 protein working solution, 2 μL test sample, then add 4 μL Tag2-DDB1/CRBN protein working solution, and 10 μL mixed Antibody Mix sequentially into a 384-well shallow plate.

2.2 Minimum Control Wells: Use 2 μL 1× Detection buffer in place of the test sample.

2.3 Maximum Control Wells: Use 2 μL 40000 nM PROTAC IRAK4 DEGRADER-1 in place of the test sample.

2.4 NC (Eu only): Add 16 μL 1× Detection buffer + 4 μL diluted Anti-Tag1 Eu dye antibody.

Table 4. Plate Layout and Controls

*For negative controls and NC, it is recommended to use matrix-matched solution instead of 1× Detection buffer.

3. Detection

Read on a TR-FRET-compatible plate reader (excitation 320 nm, emission detection at 620 nm and 665 nm).

[Data Analysis]

Calculate Signal Value:
Signal Value = (665 nm fluorescence / 620 nm fluorescence) × 10,000

Calculate Net Signal:
Net Signal = (Std – NC) / NC × 100

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

[Example Data]

The following data are for illustrative purposes only and cannot replace data obtained from your experiment. Results may vary depending on the TR-FRET-compatible instrument used.