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UniOne® TR-FRET Human CDK2/CRBN PROTAC Binding Kit

UniOne® TR-FRET Human CDK2/CRBN PROTAC Binding Kit

Catalog Number: UA086128 Brand: UA BIOSCIENCE
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Regular price $730 USD
Regular price Sale price $730 USD
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Product Details

Product Specification


Host Human
Stability & Storage

-80℃

Background

The assay kit employs homogeneous time-resolved fluorescence (TR-FRET) technology to measure the interaction between the molecular glue CDK2 Degrader7 (or test compounds) and the DDB1/CRBN complex with CDK2. This method enables simple and rapid high-throughput screening of small molecules capable of mediating the interaction between the DDB1/CRBN complex and CDK2.

As shown in the figure below, the interaction between DDB1/CRBN and CDK2 is detected using a Eu-labeled anti-Tag1 antibody (TR-FRET donor) and an Ac-labeled anti-Tag2 antibody (TR-FRET acceptor). The molecular glue CDK2 Degrader7 mediates the interaction between DDB1/CRBN and CDK2, 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 CDK2 Degrader7 between DDB1/CRBN and CDK2. The homogeneous assay is straightforward and requires no washing steps.

Components

Product Components and Storage Conditions】

Component

Concentration

100T

500T

2500T

10000T

Storage Temperature

Tag1-CRBN protein

10

5μL

20μL

100μL

400μL

-80℃

Tag2-CDK2 protein

100×

5μL

20μL

100μL

400μL

-80℃

CDK2 Degrader 7

1mM

5μL

5μL

25μL

100μL

-80℃

Anti-Tag1 Eu antibody

5

5μL

50μL

250μL

1mL

-80℃

Anti-Tag2 Ac antibody

100×

5μL

25μL

125μL

500μL

-80℃

Detection buffer

10×

400μL

2mL

10mL

40mL

-80℃


Note: All components should be aliquoted immediately after the first thaw and stored at the recommended temperature to avoid dilution storage and repeated freeze-thaw cycles.

Protocol

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1. Reagent Preparation

1.1 Thaw all reagents to room temperature before use (equilibrate for at least 30 min). The reaction system for 384-well shallow plates is 20 μL (reagent volumes are shown in the table below). Calculate the required volume before preparation. The following preparation is for reference only, using 500 reactions as an example.

Table 1. Reagent Preparation

Reagent Name

Preparation

Volume per Well (μL)

Detection buffer

Add 2 mL of 10× Detection buffer to 18 mL of deionized water, dilute to 1×, and mix well.

-

CDK2 Degrader7

Dilute the compound to the target concentration with 1× Detection buffer according to the reaction system.

2

Tag1-CRBN protein

Take 20 μL of Tag1-CRBN protein stock solution, dilute to 2 mL with 1× Detection buffer, and mix well.

4

Tag2-CDK2 protein

Take 20 μL of Tag2-CDK2 protein stock solution, dilute to 2 mL with 1× Detection buffer, and mix well.

4

Antibody Mix

Take 50 μL of Anti-Tag1 Eu antibody stock solution, add 2.450 mL of 1× Detection buffer, mix well; take 25 μL of Anti-Tag2 Ac antibody stock solution, add 2.475 mL of 1× Detection buffer, mix well; mix the two 1:1 to prepare the Antibody Mix.

10


1.2 Gradient Dilution of Test Samples

Using CDK2 Degrader7 as an example, the diluent is 1× Detection buffer. To minimize matrix interference, it is recommended to use a solution with the same matrix as the test sample for dilution. Adjust the dilution based on the actual concentration of the sample.

Table 2. Gradient Dilution of CDK2 Degrader7 (adjust as needed)

CDK2 Degrader7

Final Concentration (nM)

CDK2 Degrader7 Preparation Concentration (nM)

Preparation Method

1000

10000

1 μL of 1 mM CDK2 Degrader7 + 99 μL of 1× Detection buffer

333.3333

3333.333

20 μL of ① + 40 μL of 1× Detection buffer

111.1111

1111.111

20 μL of ② + 40 μL of 1× Detection buffer

37.0370

370.370

20 μL of ③ + 40 μL of 1× Detection buffer

12.3457

123.457

20 μL of ④ + 40 μL of 1× Detection buffer

4.1152

41.152极u>

20 μL of ⑤ + 40 μL of 1× Detection buffer

1.3717

13.717

20 μL of ⑥ + 40 μL of 1× Detection buffer

0.4572

4.572

20 μL of ⑦ + 40 μL of 1× Detection buffer

0.1524

1.524

20 μL of ⑧ + 40 μL of 1× Detection buffer

Blank

0

0

40 μL of 1× Detection buffer


2. Sample Loading and Controls

2.1 Test samples: Add 4 μL of Tag1-CRBN protein working solution, 4 μL of Tag2-CDK2 protein working solution, 2 μL of gradient-diluted test sample, and 10 μL of mixed Antibody Mix sequentially into the 384-well shallow plate.

2.2 Positive control standard curve: Add 4 μL of Tag1-CRBN protein working solution, 4 μL of Tag2-CDK2 protein working solution, 2 μL of gradient-diluted CDK2 Degrader7, and 10 μL of Antibody Mix.

2.3 Blank control wells: Replace the test sample with 2 μL of 1× Detection buffer.

2.4 NC: Add 10 μL of 1× Detection buffer and 10 μL of Antibody Mix.

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

Positive Control Standard Curve

Test Samples

Blank Control Wells

NC

2 μL

Gradient-diluted CDK2 Degrader7

2 μL

Gradient-diluted test sample

2 μL

1× Detection buffer

10 μL of 1× Detection buffer

plus 10 μL of Antibody Mix

4 μL of Tag1-CRBN protein

4 μL of Tag2-CDK2 protein

10 μL of Antibody Mix

Seal the plate with sealing film, and incubate at room temperature for 2 hours.


3. Detection

Measure using a TR-FRET-compatible microplate reader. Excitation wavelength is 320/340 nm, and emission wavelengths are 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) × 10,000

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 experimental results and is provided as an example only. Results may vary depending on the plate reader used.  

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

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