KeyTec® TR-FRET CDKs Kinase Assay Kit
The ultimate solution for cost control, superior performance, and high efficiency—all in one! Achieve an ultra-high signal-to-noise ratio with minimal enzyme usage. This time, you can have the best of both worlds!
In the field of kinase activity detection, there are two main types of assay methods:
1. Measuring the amount of phosphorylated substrate generated by kinase catalysis (e.g., Revvity’s HTRF® KinEASE™ Assay and LANCE® Ultra Kinase Assay).
2. Detecting ADP production during the kinase reaction (e.g., Promega’s ADP-Glo™ Kinase Assay).
Among these, the TR-FRET-based method for detecting phosphorylated substrates has become the widely accepted "gold standard" for kinase activity assays due to its high sensitivity, specificity, ease of operation, and stable signal performance.
For a long time, kinase assay kits were monopolized by foreign companies. However, VKEY-BIO has now broken this monopoly by launching the first domestically developed kinase activity assay kit based on KeyTec® TR-FRET technology. Let’s take a closer look at the features of the KeyTec® TR-FRET CDKs Kinase Assay Kit.
✔ Optimized Fluorescent Acceptor-Labeled Substrate (Rb/ATF2-LA):
VKEY-BIO’s KeyTec® TR-FRET acceptor fluorophore LA is covalently linked to the kinase substrate, enhancing detection sensitivity and eliminating the need to optimize the substrate-to-streptavidin (SA) ratio, making the assay simpler and more convenient.
✔ Ultra-Low Enzyme Consumption:
The kinase reaction requires as little as 0.02 ng/well of enzyme, significantly reducing experimental costs.
✔ Comprehensive Solution:
Kinases and assay kits are sold together, avoiding post-sales disputes.
✔ Compatible with Kinase Profiling:
Covers all CDKs kinase types with fully optimized reaction conditions, ensuring consistency in reaction time and temperature across different kinases.
✔ TCEP Replaces DTT:
Eliminates harmful odors and improves reaction stability.
The cell cycle is a fundamental process in cellular life activities, regulating the transition from quiescence to proliferation. Cyclin-dependent kinases (CDKs) and cyclins play a central role in this complex regulatory mechanism. This discovery was first made by Tim Hunt, Paul Nurse, and Leland H. Hartwell, who were awarded the 2001 Nobel Prize in Physiology or Medicine for their groundbreaking work.
Dysregulation of the cell cycle is a hallmark of many human cancers, and CDK inhibitors have shown great potential in this field, playing a crucial role in cancer therapy. Currently, the U.S. FDA has approved several CDK4/6 inhibitors, including Pfizer’s palbociclib, Novartis’ ribociclib, and Eli Lilly’s abemaciclib, all of which are used as first-line treatments for advanced breast cancer.
The KeyTec® TR-FRET CDKs Kinase Assay Kit measures the relative activity levels of kinases using a CDKs-specific substrate and detection reagents.
The CDK family includes multiple members:
- CDK1/3/4/6/7/9/10/12/13, 15-18 specifically phosphorylate Serine 780 (S780) of the Retinoblastoma-associated protein (Rb).
- CDK2/5 specifically phosphorylate Threonine 71 (T71) of Activating Transcription Factor 2 (ATF-2).
Thus, this kit uses optimized Rb (S780)/ATF2 (T71) peptides as substrates, directly conjugated with KeyTec® TR-FRET LA*¹, along with a phospho-specific antibody labeled with KeyTec® TR-FRET Solar Eu*². By detecting phosphorylated substrate levels, kinase activity can be assessed.
This kit employs a sandwich immunoassay based on KeyTec® TR-FRET technology, offering high specificity, sensitivity, and reproducibility. The workflow is as follows (Figure 1):
1. Kinase Reaction: The kinase phosphorylates the substrate in the presence of ATP.
2. Detection: EDTA stops the reaction, and a phospho-specific antibody is added. When the antibody binds to the phosphorylated substrate, TR-FRET donor (Solar Eu) and acceptor (LA) come into proximity, generating a TR-FRET signal upon excitation. The signal intensity is proportional to the amount of phosphorylated substrate.
*¹ KeyTec® TR-FRET LA: TR-FRET Acceptor
*² KeyTec® TR-FRET Solar Eu: TR-FRET Donor
Figure 1. KeyTec® TR-FRET Kinase Activity Detection Principle
Reaction Setup
Reaction Components | Total Volume (20 µL) | |
Enzyme Reaction Phase （10 µL） | Sample or Positive Control Compound | 2 µL |
Kinase | 4 µL | |
Rb(S780) /ATF2（T71）substrate - LA | 4 µL (premix) | |
ATP | ||
Detection Phase（10 µL） | p-Rb(S780)/ATF2（T71） mAb - Solar Eu | 10 µL |
1、CDKs Competitive Assay Comparison (CDK4/CycD3 Example)
Figure 2. VKEY-BIO vs. Competitor CDK4 Assay Results
Key Findings:
✔ Under shorter reaction time and 1/10th the enzyme amount, VKEY-BIO’s assay shows higher sensitivity (3×better) and a larger detection window than imported brands.
✔ Under the same conditions, VKEY-BIO’s CDK4/CycD3 activity matches competitor performance.
✔ Combining VKEY-BIO’s kinase and assay kit yields superior results with lower enzyme usage and shorter reaction time.
2. Kinase Assay Data (CDK2/5/4/6)
Figure 3. CDK2 Assay Data
Figure 4. CDK5 Assay Data
Figure 5. CDK4 Assay Data
Figure 6. CDK6 Assay Data
* For detailed experimental conditions and full data, refer to the manual. Contact customer service via our official WeChat account.
CAT. | Description | Size |
A1080006S | KeyTec® TR-FRET CDKs kinase kit - Rb(S780) substrate | 1,000 tests |
A1080006L | KeyTec® TR-FRET CDKs kinase kit - Rb(S780) substrate | 20,000 tests |
A1080006B | KeyTec® TR-FRET CDKs kinase kit - Rb(S780) substrate | 100,000 tests |
A1080007S | KeyTec® TR-FRET CDKs kinase kit - ATF2(T71) substrate | 1,000 tests |
A1080007L | KeyTec® TR-FRET CDKs kinase kit - ATF2(T71) substrate | 20,000 tests |
A1080007B | KeyTec® TR-FRET CDKs kinase kit - ATF2(T71) substrate | 100,000 tests |
Table 1. VKEY-BIO Kinase Assay Kit Catalog
Table 2. VKEY-BIO Kinase Product List
We have optimized reaction conditions and data for all CDKs kinases. The kinases used in experiments (Table 2) can be ordered directly by catalog number. For free trial kits or additional data, follow the VKEY-BIO WeChat official account and submit your request. Our team will contact you promptly.
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