Cisbio Bioassays and Millipore have combined their expertise to design kits for profiling and HTS of a large panel of kinases. To date, more than 160 kinases have been validated.
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.
The basic assay involves two steps:
Step 1: Kinase reaction
The kinase is incubated in the presence or absence of compounds and the appropriate substrate. The supplemented enzymatic buffer is added in the kinase buffer for tyr kinases only. ATP is added to start the reaction.
Step 2: Detection
Eu3+ Cryptate and XL665 conjugates are added. The detection buffer contains EDTA to stop the enzymatic reaction.
The HTRF KinEASE kits use the same assay format. Each kit includes a universal biotinylated substrate, a monoclonal phosphor-specific antibody labeled with Eu3+-Cryptate labeled antibody, SA-XL665 and assay buffers. The KinEASE platform consists of a choice of three different kits with different substrates for Ser/Thr kinase assays (KinEASE STK S1, S2 and S3), and one kit for Tyr kinase assay (KinEASE TK). All kits are available in Bulk and Jumbo sizes for HTS and profiling. A Ser/Thr Discovery kit, including all 3 substrates, is also available to determine the optimal setup for each assay. A proprietary substrate stabilizing buffer (SEB) to be used with KinEASE TK enhances assay performance while maintaining inhibitor properties such as IC50s.
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) (Figure 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).
Full assay optimization was performed on MAPKAP-K2, CaMKIV, PKCbII, Rsk3, ROCK-II, Pim-1: 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 below.
Staurosporine IC50 values were determined 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.
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.
More than 160 kinases have already been validated and the optimal substrates identified.
Figure adapted from Cell Signaling Technology.
| Click below to see the list of validated kinases |
|---|
| HTRF® kinEASE STK S1 |
| HTRF® kinEASE STK S2 |
| HTRF® kinEASE STK S3 |
| HTRF® kinEASE TK |
| Description | Cat. no | Product insert | MSDS |
|---|---|---|---|
| HTRF KinEASE -STK - 1,000 tests - Discovery kit | 62ST0PEB | - | |
| HTRF KinEASE -STK S1 kit - 1,000 tests | 62ST1PEB | ||
| HTRF KinEASE -STK S1 kit - 20,000 tests | 62ST1PEC | ||
| HTRF KinEASE -STK S1 kit - 100,000 tests | 62ST1PEJ | ||
| HTRF KinEASE -STK S2 kit - 1,000 tests | 62ST2PEB | ||
| HTRF KinEASE -STK S2 kit - 20,000 tests | 62ST2PEC | ||
| HTRF KinEASE -STK S2 kit - 100,000 tests | 62ST2PEJ | ||
| HTRF KinEASE -STK S3 kit - 1,000 tests | 62ST3PEB | - | |
| HTRF KinEASE -STK S3 kit - 20,000 tests | 62ST3PEC | ||
| HTRF KinEASE -STK S3 kit - 100,000 tests | 62ST3PEJ | ||
| HTRF kinEASE-TK kit - 1,000 tests | 62TK0PEB | ||
| HTRF kinEASE-TK kit - 20,000 tests | 62TK0PEC | ||
| HTRF kinEASE-TK kit - 100,000 tests | 62TK0PEJ |
| Description | Cat. no | Product insert | MSDS |
|---|---|---|---|
| SEB - Suppl. Enzymatic Buffer - 12.5 nmoles | 61SEBALB | - | |
| STK-Substrate 1-biotin - 500 | 61ST1BLC | - | |
| STK-Substrate 1-biotin - 50 | 61ST1BLE | - | |
| STK-Substrate 2-biotin - 500 | 61ST2BLC | - | |
| STK-Substrate 2-biotin - 50 | 61ST2BLE | - | |
| STK-Substrate 3-biotin - 500 | 61ST3BLC | - | |
| STK-Substrate 3-biotin - 50 | 61ST3BLE | - | |
| TK Substrate-biotin - 500 | 61TK0BLC | - | |
| TK Substrate-biotin - 50 | 61TK0BLE | - | |
| Enzymatic buffer/kinase 5X - 10 ml | 62EZBFDC | - | |
| Enzymatic buffer kinase 5X - 50 mL | 62EZBFDD | - | |
| HTRF detection buffer - 40 ml | 62SDBRDD | - | - |
| HTRF KinEASE detection buffer - 200 mL | 62SDBRDF | - |
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