HTRF® KinEASE™ TK
Universal assay for Tyrosine Kinases
Cisbio Bioassays's HTRF® KinEASE™ platform has expanded to include a universal assay for tyrosine kinases. The HTRF KinEASE TK assay is based on our patented HTRF technology and uses a unique tyrosine peptide substrate in combination with a Europium-labeled monoclonal antibody. The HTRF KinEASE™ TK assay has been validated on 59 tyrosine kinases to-date including both receptor and cytoplasmic TKs. A proprietary substrate stabilizing buffer (SEB) enhances assay performance, enables the use of lower enzyme concentrations, while maintaining inhibitor properties such as IC50s.
- Validated on 59 Tyr kinases
- Low enzyme consumption
- Proprietary substrate stabilizing buffer
- Results in under two hours
- Easy miniaturization down to 4µL
- Receptor & Cytoplasmic tyrosine kinases
- Monitoring tyrosine kinase activity
- High throughput kinase screening
- Kinase profiling
Assay Principle:
HTRF KinEASE TK assays use a proprietary anti-phosphotyrosine specific Mab label with Eu3+ -Cryptate and a unique biotinylated TK kinase substrate detected using XL665 labeled streptavidin. The basic assay involves two steps:
Enzymatic step:
the kinase is incubated in the presence or absence of compounds, the biotinylated substrate and a supplemental enzyme buffer (SEB). ATP is added to start the reaction.
Detection step:
Eu3+ Cryptate and XL665 conjugates are added. The detection buffer contains EDTA to stop the enzymatic reaction. The two detection reagents can be pre-mixed allowing for a single reagent addition.
Product Specifications:
The HTRF® KinEASE™ TK reaction is run in the presence of a proprietary substrate stabilizing buffer. Figure 1 shows the effect of various concentrations of the SEB reagent on assay S-B. Use of this buffer greatly enhances S-B. Negative effects on assay performance are not seen even at higher concentrations (50 nM).
In addition to increasing assay S-B, SEB enables the use of lower kinase concentrations per well while maintaining large S-B. Titration of the Abl tyrosine kinase was run in tandem in the presence of 2.5 or 5 nM SEB reagent. As shown in Figure 2, 5 nM SEB enhances S-B to such a large degree as to allow lower kinase usage.
Figure 1: Abl (Millipore 14-459), used at 5 ng/well, was incubated 30 min with substrate-biotin (1 µM), and a non-limiting ATP concentration (100 µM) in a final assay volume of 20 µl. SEB concentrations ranging from 0 nM to 50 nM were tested.
Figure 2: Abl, used at concentrations ranging from 0.10 ng/well to 10 ng/well, was incubated 30 min with the substrate-biotin (1 µM), SEB (2.5 nM and 5 nM) and a non-limiting ATP concentration (100 µM).
Assay Performance:
The IC50s for a panel of tyrosine kinases were determined in the presences or absence of 50nM SEB reagent. The concentration of kinase used in the assays was optimized for each kinase.
Table 1 IC50 values for staurosporine and known kinase-specific inhibitors were determined for six kinases. All assays were performed using the standard assay protocol with or without SEB. Assays were run using Millipore kinases.
Validated Tyrosine Kinases:
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Figure 3: List of receptor and cytoplasmic tyrosine kinases validated using HTRF® KinEASE™ TK. Figure adapted from Cell Signaling Technology.
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Ordering Info:
HTRF® KinEASE™ TK Reagents:
| Description | Quantity* | Cat no. | Product Insert |
| HTRF® KinEASE™ TK | 1,000 tests 20,000 tests 100,000 tests |
62TK0PEB 62TK0PEC 62TK0PEJ |
pdf |
Companion Products:
| Description | Quantity* | Cat no. |
| Sa-XL665 | 250µg 1 mg |
610SAXLA 610SAXLB |
| TK substrate-biotin | 50µg/vial 500µg/vial |
61TK0BLE 61TK0BLC |
| HTRF Detection buffer | 200mL | 62SDBRDF |
| Sa-XL665 | 3mg | 610SAXLG |
For more information or To Place an Order:
U.S. and Canada
1-888-963-4567
Europe and other countries
+33(0)466 796 705
Japan
+81-3-5510-2932
