HTRF® KinEASE™
Universal Assay for Serine/Threonine and Tyrosine Kinases
HTRF KinEASE® is a universal platform for straightforward Serine/Threonine (STK) and tyrosine (TK) kinase screening, based on our patented HTRF® technology. It consists of a choice of four kits, each including a proprietary monoclonal antibody and a universal biotinylated peptide developed by Millipore Corporation. KinEASE kits use the same assay format and provide additional benefits such as low enzyme consumption and compatibility with a wide range of ATP concentrations. A proprietary substrate stabilizing buffer (SEB) to be used with KinEASE TK enhances assay performance while maintaining inhibitor properties such as IC50s. To date, more than 160 kinases have been validated.
- Validated on over 107 Ser/Thr and 58 Tyr kinases
- Low enzyme consumption
- Highly sensitive assays
- Broad range of ATP (up to mM)
- Proprietary substrate stabilizing buffer for Tyr (SEB)
- Easy miniaturization down to 4µL
- Ser/Thr and Tyr kinase monitoring
- High throughput kinase screening
- Kinase selectivity and profiling
- Mechanistic studies (MOA)
Assay Principle:
HTRF® KinEASE™ kits use a proprietary phospho-specific monoclonal antibody 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. The supplemented enzymatic is added in the kinase buffer for tyr kinases only . 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.
Product Specifications:
Four kits, HTRF® KinEASE™ STK S1, S2 and S3 and TK, each containing a single biotinylated substrate, the Eu3+-Cryptate labeled antibody, SA-XL665 and assay buffers.
Table 2 (below) summarizes the optimal substrates identified by Cisbio Bioassays for over 160 Ser/Thr and Tyr kinases, and allows researchers to order the appropriate HTRF KinEASE kit. The kits are available in Bulk and Jumbo sizes for HTS and profiling.
HTRF® kinEASE™ STK discovery kit includes all three biotinylated STK substrates for use in identifying the optimum substrate for your specific Ser/Thr kinases. This kit is particularly useful in assay development. As shown in Figure 1, STK substrate 3 (S3), which produced the largest S/B, was the preferred substrate for MST1 kinase assay.
Figure 1: Various concentrations of MST1 were used in combination with S1, S2 and S3. Standard HTRF® KinEASE™ STK conditions and protocols were used.
The HTRF® KinEASE™ TK reaction is run in the presence of a proprietary substrate stabilizing buffer. This buffer greatly enhances S-B and enables the use of lower kinase concentrations per well. Titration of the Abl tyrosine kinase was run in tandem in the presence of 2.5 or 5 nM of SEB reagent. As shown in Figure 2, 5 nM SEB enhances S-B so much that lower kinase usage is possible. The use of SEB does not affect assay performances, even at high concentrations (up to 50 nM) (fig.3).
Figure 2: Abl (Millipore Corporation 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 3: 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:
Over 107 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: 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 and ATP KM concentrations, with 30 min incubation in a 20 µl final assay volume in low volume 384-well plates, followed by the addition of Eu3+ Cryptate labeled antibody and SA-XL665. Plates were incubated for 1 hour moe, and the signal read at 620 and 665nm.
The inhibitor IC50s for a panel of tyrosine kinases were determined using KinEASE TK and following the standard assay protocol in the presence or absence of 50nM SEB reagent. The kinase concentration used in the assays was optimized for each.
Table 1: IC50 values for staurosporine and known kinase-specific inhibitors were determined for six Tyr kinases (Millipore Corporation) in 20µL final 384w plate.
Validated kinases:
More than 160 kinases have already been validated and the optimal substrate identified using Millipore Corporation kinases (table 2). Figure adapted from Cell Signaling Technology.
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Ordering Info:
HTRF® KinEASE™ 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 20,000 tests 100,000 tests |
62ST1PEB 62ST1PEC 62ST1PEJ |
pdf |
| HTRF® KinEASE™ STK S2 (STK substrate2-biotin) |
1,000 tests 20,000 tests 100,000 tests |
62ST2PEB 62ST2PEC 62ST2PEJ |
pdf |
| HTRF® KinEASE™ STK S3 (STK substrate3-biotin) |
1,000 tests 20,000 tests 100,000 tests |
62ST3PEB 62ST3PEC 62ST3PEJ |
pdf |
| HTRF® KinEASE™ TK (TK substrate-biotin) |
1,000 tests 20,000 tests 100,000 tests |
62TK0PEB 62TK0PEC 62TK0PEJ |
pdf |
HTRF KinEASE 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 |
| TK substrate- biotin | 500µg/vial | 61TK0BLC |
| Sa-XL665 | 250µg 1mg |
610SAXLA 610SAXLB |
| Enzymatic Buffer/kinase 5x | 50 mL | 62EZBFDD |
| HTRF® Detection buffer | 200mL | 62SDBRDF |
| SEB buffer for KinEASE TK assays | 20,000 tests (lyoph, to be reconst. with 5 ml H2O) |
61SEBALB |
