Which event should my kinase assay measure?
ADP production, ATP depletion, substrate phosphorylation and target binding are useful measurements. They are not interchangeable evidence of kinase inhibition.
Discovery in Practice · Reference library
Assays
Choose and interpret assay formats by the biological event and detection sequence.
The same signal change can have several causes. Find the assay you use, then follow its controls and the relevant method comparison.
Start with these questions
Do I need DNA mass, target copies or usable library molecules?
DNA mass, target copies, amplifiable library molecules and fragment integrity are different measurements. Choose the answer your next experiment needs before choosing the instrument.
More in this topic · newest first
Can tracer linker length change apparent binding?
A one-carbon linker change can alter tracer affinity and assay performance. Learn how to separate binding chemistry from your fluorescence polarization window.
Which two measurements are hidden by an FP ratio?
A fluorescence-polarization value is calculated from parallel and perpendicular intensity measurements, and the ratio can hide low photon counts or timing mismatch. The article explains how to preserve both channels, interpret intensity and binding curves, and qualify tracer and reader behavior.
Did the tagged protein disappear, or just the HiBiT signal?
A decrease in HiBiT light can reflect less tagged protein or a change in complementation, luciferase activity, lysis, background, or measurement. The article separates reporter abundance from accelerated degradation and outlines controls that test the detection system and native protein.
Does less CellTiter-Glo light mean fewer cells?
CellTiter-Glo measures ATP-dependent light, not cell number directly, so metabolism and detection conditions can change the signal independently of abundance. The article separates biological effects from reagent, temperature, mixing, and detector effects and proposes controls for each.
Did kinase activity change, or did ADP-Glo detection change?
ADP-Glo light is the endpoint of a multistep detection sequence, so a lower signal can arise outside the kinase reaction. This article shows how to reconstruct the endpoint with ATP/ADP controls and interpret temperature, timing, conversion, and ATP-dependent potency.
What can a stable HTRF ratio hide?
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.