What does the spatial pattern of wells tell me?
Inspect spatial patterns alongside control statistics, then use those patterns to choose a follow-up check.
Discovery in Practice · Reference library
Troubleshooting
Practical questions about unexpected signals, interference and assay precision.
Choose the symptom closest to your result. Each link leads to a full article with the assumptions and checks needed to interpret it.
Start with these questions
Can a better Z′ conceal a nonlinear measurement?
A higher Z′ can reflect nonlinear detector compression rather than a better assay. This article explains how bright-well variation can shrink while effect sizes become distorted, and how to check proportionality without confusing detector behavior with assay chemistry.
More in this topic · newest first
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.
When does a longer luminescence read stop helping?
Longer luminescence reads help when photon collection limits precision, but they cannot remove persistent differences between wells. This article shows how to compare repeated reads with independently prepared replicates, account for background noise, and choose a useful stopping point.