SKU: 47002108700

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

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Description

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

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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4.6 ★★★★★
Based on 24 reviews
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M
Verified Purchase
M
Houston, US
★★★★★ 4
Not for gnawers
Size: 11.5 in, Color: Bear
I was hoping this would work for my teenage ACD and save my leather goods from being chewed on. He gnawed on it quietly I was thrilled. But I had to take it from him within a few minutes. Gnawing apparently equals chewing pieces off. Not made for chewers or gnawers. It seemed to be well made, was soft, was actually a fairly good size and didn’t get gross when it got spitty. Just didn’t work out for Captain chaos destructor of all.
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Reviewed in the United States on May 4, 2026
M
Verified Purchase
Maria Peters
Massapequa, US
★★★★★ 5
Durable & Leather. For Small-Medium Dogs
Size: 15 in, Color: Retriever, Size: 15 in, Color: Retriever
This is a great dog toy! My Pomeranian is a small guy at only 8lbs. but he sure knows how to chew and he does it as often as he can. I was throwing out toy after toy because he was tearing them to pieces after only a half hour or so. After putting my money in the garbage so many times, I decided to search for toys that would hold up to his destructive mouth. I found a lot that claimed they wouldn't fall apart but they were all more than I wanted to spend. I came across this toy and a rabbit shaped one just like this. I bought both months ago and both are still in tact after all this time. Sure the edges are a little frayed but other than that, no damage whatsoever! I'm not sure what dura fused means but that seemed promising to me when I was reading the description, also the fact that they're leather made them seem more durable to me. Both have squeakers inside and some sort of stuffing as well. I throw all of my dogs toys in the laundry or hand wash them once a week, except for these. When these toys get wet with my dogs saliva, they look like they're not meant to get wet. Even without washing, these seem fine. This toy is probably about 9" long or so if I had to guess. In my opinion, it'd be perfect for a medium sized dog. It's a tiny bit big for my little guy but he manages just fine! If you're looking for a durable toy that your small to medium dog can chew on constantly, look no further!
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Reviewed in the United States on February 3, 2020
G
Verified Purchase
GK5040
Bozeman, US
★★★★★ 5
Always looking for less destructible dog toys
Size: 8 in, Color: Squirrel, Size: 8 in, Color: Squirrel
I am constantly searching for aggressive chewer dog toys. So many claim to be and one look and you know they haven’t met your dog!! Our golden retriever is so sweet until he gets his claws and teeth into a new toy. We nicknamed him the shredder, as he rips his new toys to pieces. I accidentally stumbled across this toy. We have never owned a leather dog toy. The squirrel is going to give Maverick a run for his money. The edges are double sewn closed. I am very impressed with the quality of this toy for $5.75 which includes the sales price and an additional 10% off coupon. Even without those two incentives the toy is less than $8 prime. The other shapes are similar or cheaper in price and now I see a 50% off 1 toy coupon when you buy 2. What a deal and the squirrel has a squeaker inside. What I like about the squirrel is that there’s less limbs or appendages for Maverick to shred off. The bunny has ears, the raccoon has hands, the buffalo has legs, the bone and retriever stick have a rope, the bear has a neck and head that could be chewed off, similar to the fox who has a tail and snout. The squirrel looks like it would be harder to destroy! The squeaker was hard to push at first but is easier now. Maverick has a black lab sister who loves squirrels, more so than she loves toys, and I found this toy searching for squirrel toys for her. It is offered in 8 other shapes. The squirrel measures almost 8” tall by 5” wide. It’s perfect for a medium or large size dog. Based on the construction of this squirrel dog toy it looks acceptable for an aggressive chewer and won’t break the bank. It’s very affordable. Always supervise! Updates to follow at Christmas:). BTW, the squirrel did not smell bad upon opening.
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Reviewed in the United States on December 3, 2024
C
Verified Purchase
Cakirsch
Belleville, US
★★★★★ 4
Puppy loves it. Well built and fun for her to play with.
Size: 15 in, Color: Retriever
My double doodle mix loved this toy. It is well made and she loves the different texture of the rope. Sadly she has already torn the rope apart and so now it’s just the leather part. This would be the perfect toy for non agressive chewers. Wish the rope lasted a little longer. SPOT is my new favorite company to get her chew toys from now. They’re well built and aren’t expensive like all the other dog toys out there.
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Reviewed in the United States on June 30, 2025
K
Verified Purchase
KAV
Los Angeles, US
★★★★★ 2
Destroyed in under 10 minutes
Size: 9 in, Color: Large Bone
I bought 2 of these toys, one for my 9 year old great dane who isn't an aggressive chewer, and one for my 6 month old puppy who is average. In less than 10 minutes both were destroyed and my dane managed to swallow a good chunk. I wasn't surprised when they both had small holes on the ends and started pulling the stuffing out. The stuffing and squeakers were confiscated immediately, but I let them keep chewing. By the time I came back from throwing the insides away, both dogs had chewed the toys in half and my dane swallowed a chunk when he saw me coming. The puppy was going to do the same thing, but luckily we're working on his "drop" command so he was excited enough for the bigger payout and dropped it. They didn't just come apart at the seams, the whole toy ripped in half making it too easy to swallow. Neither of them are aggressive cheers and the toys didn't hold up at all. A stuffed animal toy would have lasted longer (and has any time I've allowed it). 10 minutes of playtime is probably too generous , but I'm trying to be fair. If I hadn't been right there watching them, they both would have been able to swallow the whole thing in minutes. Definitely not safe for any amount of actual chewing.
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Reviewed in the United States on November 14, 2025

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