Interpret time-resolved fluorescence ratios and choose assay and acquisition controls.
Begin with both emission channels, then connect the readout to the biochemical event the assay is intended to report.
A stable HTRF ratio can conceal a major loss of signal, degraded precision, or a channel-specific interference. The article explains how to retain and interpret both emission channels, per-well ratios, and acquisition conditions.
ADP production, ATP depletion, substrate phosphorylation and target binding are useful measurements. They are not interchangeable evidence of kinase inhibition.