Product Details
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 |
100× |
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 |
50× |
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 |
① |
1000 |
10000 |
1 μL of 1 mM CDK2 Degrader7 + 99 μL of 1× Detection buffer |
② |
333.3333 |
3333.333 |
20 μL of ① + 40 μL of 1× Detection buffer |
③ |
111.1111 |
1111.111 |
20 μL of ② + 40 μL of 1× Detection buffer |
④ |
37.0370 |
370.370 |
20 μL of ③ + 40 μL of 1× Detection buffer |
⑤ |
12.3457 |
123.457 |
20 μL of ④ + 40 μL of 1× Detection buffer |
⑥ |
4.1152 |
41.152极u> |
20 μL of ⑤ + 40 μL of 1× Detection buffer |
⑦ |
1.3717 |
13.717 |
20 μL of ⑥ + 40 μL of 1× Detection buffer |
⑧ |
0.4572 |
4.572 |
20 μL of ⑦ + 40 μL of 1× Detection buffer |
⑨ |
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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