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

【ASSAY PRINCIPLE】

The kit is based on homogeneous time-resolved fluorescence technology (TR-FRET) technology，which is used to assess the molecular-mediated interaction between the DDB1/CRBN complex and ITK. This method provides a high-throughput approach to detect the small molecules capable of mediating the interaction between the DDB1/CRBN complex and ITK simply and rapidly.

As shown in the figure below, the interaction between DDB1/CRBN and ITK was detected by an Eu-labeled anti-Tag1 antibody (TR-FRET donor) and an Ac-labeled anti-Tag2 antibody (TR-FRET receptor). Since the molecular adhesive BSJ-05-037 mediates the interaction between DDB1/CRBN and ITK, the donor antibody is in close proximity to the receptor antibody. Excitation of the donor antibody triggers fluorescence resonance energy transfer (FRET) to the receptor antibody, resulting in specific emission of a signal at a wavelength of 665 nm from the receptor antibody. This specific signal is proportional to the degree of interaction between BSJ-05-037, DDB1/CRBN and ITK. This homogeneous assay is simple to perform and requires no washing steps.

【Composition and Storage Conditions】

Note: Aliquot into appropriate volumes, store at the recommended temperature and avoid repeated freeze-thaw cycle.

[Process and Operations]

1, Reagent Preparation

1.1Compositionsequilibration

Before use, dissolve all reagents at room temperature (allow at least 30 minutes for temperature equilibration). For the 384-well plate assay, use 20 μL of reaction mixture (the reagent quantities are listed in the table below). Calculate the required volume prior to preparation accordingly. The following preparation steps are provided for reference only, using a 500-well plate as an example.

Table 1. Reagent Preparation

1.2Gradient dilution of the sample

BSJ-05-037as an example, the dilution solution is 1× Detection buffer. To minimize interference from matrix effects, it is recommended to use a solution with the same matrix composition as the sample being tested; additionally, the sample concentration should be adjusted according to its actual situation.

Table2.BSJ-05-037gradient dilution(Adjust according to the actual situation)

2, Sample and control

2.1Sample:Add 4 μL of Tag1-CRBN protein working solution, 4 μL of Tag2-ITKprotein working solution, 2 μL of the gradient-diluted sample , and 10 μL of the mixed Antibody Mix sequentially into the 384-well plate.

2.2BSJ-05-037:4 μL of Tag1-CRBN protein working solution, 4 μL of Tag2-ITKprotein working solution, 2 μL of gradient-dilutedBSJ-05-037, and 10 μL of the mixed Antibody Mix sequentially into the 384-well plate.

2.3 Blank:4 μL of Tag1-CRBN protein working solution, 4 μL of Tag2-ITKprotein working solution, 2 μL ofDetection buffer, and 10 μL of the mixed Antibody Mix sequentially into the 384-well plate.

2.3Negative control:10μL Detection buffer, and 10 μL of the mixed Antibody Mix sequentially into the 384-well plate.

After adding all samples, centrifuge, seal with a cover membrane, and incubate at room temperature for 2 hours.

Table 3.Sample and control spotting process

3,Read Data

The detection was performed on a TR-FRET microplate reader. The excitation wavelength was 320/340 nm, and the emission wavelengths at 620 nm and 665 nm were detected.

[DATA REDUCTION]

1) Ratio:

Ratio = (665/620)×10000

2) Net signal:

Net signal= (Std-NC)/NC×100

Std: the ratio of sample and control

NC: the ratio ofnegative control

3)CV (%):

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

[Data Example]

The following data cannot replace experimental results obtained in experiments;theyare provided solely for illustrative purposes, and outcomes may vary depending on the plate reader used. 

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