Figure 11 Tyrosine kinase assay using HTRF reagents. Biotinylated substrate binds streptavidin-XL665 and antiphosphotyrosine-(Eu)K recognizes those peptides that are phosphorylated. Figure 12 Titration of kinase inhibitor for src-1 family of enzymes.

Sensitivity is a critical issue in HTS when the cost of reagents must be considered and the availability of enzyme and sample materials is limited. In order to determine sensitivity of the HTRF chemistry for kinase activity, varying concentrations of Ick enzyme was added reactions containing peptide and ATP. At time points up to 80 min reactions were stopped and measured in the Discovery. As little as 20 pM Ick enzyme gave significant results with a signal-to-noise background of 10:1 after 40 min (Fig. 13). This result demonstrates that the HTRF assay method provided about two orders of magnitude more sensitivity than the SPA assay method, making it the first nonradioisotopic alternative capable of yielding results that could be achieved previously only with the use of radioisotopes. The