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| Kinasera-TRF assay |
Luminescence is emitted only when the ARC-Lum probe is bound to protein kinase (PK). Displacement of the probe from the complex by a competitive inhibitor (C) leads to disappearance of the luminescence signal. Therefore the detected signal is directly proportional to the amount of the ARC-Lum:Kinase complex in the solution. The luminescence signal is not affected by the presence of unbound ARC-Lum probe.
As unbound ARC-Lum probe does not substantially contribute to TRF signal, high concentrations of ARC-Lum probe (up to 100 nM) can be used in the assay for the determination of binding affinities of compounds that under conventional assay conditions demonstrate tight binding properties (eg., Ki values of Staurosporine towards several kinases is less than 1 nM and cannot be precisely determined with substrate phosphorylation based assays).
Kinasera-TRF assay can be easily adapted for the analysis of other basophilic members of AGC group of protein kinases. As ARC-Lum can be used in excess, still low concentrations of kinase can be used for the assay even if the KD value for ARC-Lum:kinase complex is not in the low nanomolar range.
Kinasera-TRF assay is applicable with a wide range of TRF based detection systems including those, used for europium-based and terbium-based LanthaScreenTM and HTRF assays (excitation at 340 nm, emission at 665 nm or 520 nm respectively).