HTRF® KinEASE™

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.

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.

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.