Protein kinases (EC 2 7 10/EC 2 7 11) which phosphorylate the hyd

Protein kinases (EC 2.7.10/EC 2.7.11) which phosphorylate the hydroxyl group present on serine, threonine, or tyrosine residues represent an enzyme family for which many assays have been developed due to their central role in controlling signaling pathways (Glickman et al., 2004). Measurement of substrate depletion by detecting the remaining ATP in a kinase assay using firefly luciferase (EC is an example

of a generic assay format for protein kinases (Koresawa and Okabe, 2004 and Singh et al., 2004). The use of a bioluminescent signal for ATP levels results in an increase in luminescence when the kinase is inhibited. Drawbacks of the see more ATP depletion method include the need to run the assay at high substrate turnover which often requires larger amounts of enzyme, the assay is performed using check details single endpoint, and the assay requires the presence of a luciferase coupling enzyme. This latter point requires that appropriate counter-screens against luciferase alone are performed (Auld et al., 2008). Additionally, as the system measures substrate depletion, the standard steady-state assumption that So~St does not apply, thus confounding MoI studies. The assay is best performed using 50% conversion of substrate where the signal:background ratio is ~2-fold, a range often yielding acceptable assay performance for luminescent assays, and the expected shift in IC50 from ideal initial rate conditions is expected to be <2-fold

( Wu et al., 2003). A more desirable method to measure enzyme activity is by detecting product formation. Recently, generic methods for kinase assays have been developed that detect the ADP product. These include both a non-antibody based system that employs coupling enzymes with a fluorescent readout (DiscoveRx, ADP Quest™; λex=530 nm, λem=590 nm) ( Charter et al., 2006) and a system that uses an ADP specific antibody and red-shifted FP by employing an Alexa® 633-labeled ADP (BellBrooks Lab, Transcreener™; λex=612 nm,

λem=670 nm) ( Huss et al., 2007 and Kleman-Leyer et al., 2009). The red-shifted fluorescence limits fluorescent Cyclin-dependent kinase 3 interference by compounds and the ratiometric nature of FP aides in minimizing artifacts due to liquid handling. The BellBrook system has also been adapted to a TR-FRET format employing terbium labeled ADP-antibody and fluorescein labeled ADP ( Klink et al., 2008). The TR-FRET format of this assay limits interference by faster decaying background fluorescence due to compounds or buffer components ( Comley, 2006). Indeed, with any fluorescent-based assay, a consideration of interference by fluorescent compounds ( Simeonov et al., 2008) or by the inner-filter effect ( Palmier and Van Doren, 2007) needs to be considered. Low-molecular weight (LMW) compounds present in typical chemical libraries can show a good-deal of blue fluorescent, therefore using red-shifted fluorophores for detection can reduce interference by compound fluorescence ( Simeonov et al., 2008).

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