HTRF® KinEASE™ STK
Universal Assay for Serine/Threonine Kinases
Cisbio Bioassays's line of HTRF® KinEASE™ STK kits are based on our patented HTRF® technology and can be used as a universal tool for assessing Serine/Threonine kinase activity. These assays use a proprietary antibody and a choice of three biotinylated substrates developed by Upstate (part of Millipore), to rapidly produce high quality kinase results. Our HTRF KinEASE™ STK kits have been validated on more than 104 kinases and provide additional benefits such as low enzyme consumption and compatibility with a large range of ATP concentrations.
- Validated on over 104 Ser/Thr kinases
- Low enzyme consumption
- Highly sensitive assays
- Results in under two hours
- Easy miniaturization down to 4µL
- Serine/Threonine kinase monitoring
- High throughput kinase screening
- Kinase selectivity and profiling
Assay Principle:
HTRF® KinEASE™ STK kits use a proprietary anti-phosphoserine specific MAb labeled with Eu3+ -Cryptate and a proprietary
biotinylated 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 and the appropriate substrate.
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:
Three kits, HTRF® KinEASE™ STK S1, S2 and S3, each contain a single biotinylated substrate, the Eu3+-Cryptate labeled antibody, SA-XL665 and assay buffers. Figure 3 (below) summarizes the optimal substrates identified by Cisbio Bioassays for over 104 Ser/Thr kinases and allows researchers to order the appropriate kit. These kits are also available in Bulk and Jumbo sizes for HTS and profiling.
Our HTRF® KinEASE™ STK Discovery kit includes all three biotinylated substrates for use in identifying the optimum substrate for other Ser/Thr kinases. This kit is particularly useful in assay development. Figure 1 shows an MST1 kinase assay run with all three substrates. While all 3 substrates generate results, substrate 3 (S3) produces the largest S/B ratio and would therefore be considered the preferred substrate.
Figure 1: Various concentrations of MST1 were used in combination with S1, S2 and S3. Standard HTRF® KinEASE™ STK conditions and protocols were used
Assay Performance:
Over 104 serine/threonine kinases have been validated using HTRF® KinEASE™ STK assays. In addition, 6 kinases (MAPKAP-K2, CaMKIV, PKCbII, Rsk3, ROCK-II, Pim-1) were chosen for full assay optimization. This included identification of the optimal substrate, kinase titration, enzyme reaction kinetics, substrate and ATP KM determination, optimization of SA-XL665:substrate ratio and IC50 calculations for staurosporine. Staurosporine inhibition results are shown in Figure 2. The IC50s obtained with HTRF® KinEASE™ STK were similar to the reference radioactive method. For full optimization results click here ».
Figure 2: Staurosporine IC50 values were determined for the six kinases using the identified optimal substrate. For each enzyme the kinase, substrate and ATP concentrations used were KM values determined earlier in assay optimization process. All assays were performed in a 100µl final assay volume in low volume 384-well plates. Kinase reactions were run using various concentrations of Staurosporine for 30 minutes at room temperature followed by the addition of Eu3+ Cryptate labeled antibody and SA-XL665. Plates were incubated for 1 hour and signal read at 620 and 665nm.
Validated Serine/Threonine Kinases:
 |
Figure 3. Optimal substrates identified for 104 different serine/threonine kinases. All assays were carried out using Upstate (part of Millipore) kinases. Figure adapted from Cell Signaling Technology. 
|
Ordering Info:
HTRF® KinEASE™ STK Reagents:
| Description | Quantity* | Cat no. | Product Insert |
| HTRF® KinEASE™ STK discovery (STK substrates 1,2 and 3-biotin) |
1,000 tests | 62ST0PEB | |
| HTRF® KinEASE™ STK S1 (STK substrate1-biotin) |
1,000 tests 10,000 tests 100,000 tests |
62ST1PEB 62ST1PEC 62ST1PEJ |
pdf |
| HTRF® KinEASE™ STK S2 (STK substrate2-biotin) |
1,000 tests 10,000 tests 100,000 tests |
62ST2PEB 62ST2PEC 62ST2PEJ |
pdf |
| HTRF® KinEASE™ STK S3 (STK substrate3-biotin) |
1,000 tests 10,000 tests 100,000 tests |
62ST3PEB 62ST3PEC 62ST3PEJ |
pdf |
Companion Products:
| Description | Quantity* | Cat no. |
| STK substrate 1-biotin | 50µg/vial | 61ST1BLE |
| STK substrate 2-biotin | 50µg/vial | 61ST2BLE |
| STK substrate 3-biotin | 50µg/vial | 61ST3BLE |
| Sa-XL665 | 250µg 1mg |
610SAXLA 610SAXLB |
| HTRF® KinEASE™ TK | 1,000 tests 20,000 tests 100,000 tests |
62TK0PEB 62TK0PEC 62TK0PEJ |
| Enzymatic Buffer/kinase 5x | 50 mL | 62EZBFDD |
| HTRF® Detection buffer | 200mL | 62SDBRDF |
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
