Name the biological response before the midpoint
EC50 is commonly the concentration associated with half of a defined effective response. IC50 is commonly the concentration associated with half of a defined inhibitory response. Neither is an assay-independent property: incubation time, cell system, substrate, readout, normalization, and concentration range can change the estimate.
State what the response means. “50%” may mean halfway between fitted asymptotes (a relative midpoint) or a fixed normalized response such as exactly 50% inhibition (an absolute threshold). These values need not coincide.
| Term | Typical response question | Report with |
|---|---|---|
| EC50 | Which concentration reaches half of a defined effect? | Endpoint, model, units, fitted range |
| IC50 | Which concentration reaches half of a defined inhibition? | Normalization controls, model, units, fitted range |
| Relative midpoint | Where is halfway between fitted top and bottom? | Both fitted asymptotes |
| Absolute 50% | Where does the curve cross a fixed 50% response? | Normalization and crossing definition |
Normalization makes the denominator part of the result
A common inhibition calculation uses positive and negative controls to map raw response onto a percentage scale. If those controls drift, the normalized curve and midpoint can move even when test wells do not.
Retain raw response, control values, formula, and normalized response. Inspect control variability and plate position before treating a normalized value as ground truth. Avoid clipping values above 100% or below 0% unless the method defines and justifies that transformation.
Worked comparison: same concentrations, different questions
Suppose a stimulatory curve rises from 5% to 95% and its fitted relative midpoint is 12 nM. That result may be reported as EC50 = 12 nM. In a separate inhibitory assay, response falls from 98% activity to 8% activity with a fitted relative midpoint of 27 nM; it may be reported as IC50 = 27 nM.
The numbers cannot be ranked as if they came from the same assay. Even within one assay, compare values only when response definitions, conditions, model settings, and units are compatible. Log-transform midpoint estimates for many between-run analyses because concentration ratios are often more meaningful than raw differences.
Before reporting EC50 or IC50
Confirm that the midpoint is interpolated within the tested concentration range and the curve contains informative response on both sides. Check fitted asymptotes, residual patterns, parameter uncertainty, replicate variability, control behavior, and any method-specific criteria.
If the response does not reach a plateau or the midpoint is outside the range, an estimate may be weakly supported even when software returns a number. Report censoring or inability to estimate according to the method rather than presenting false precision.
Frequently asked questions
Is a lower IC50 always a better compound?
A lower IC50 indicates greater apparent potency under that assay’s conditions, but efficacy, selectivity, mechanism, exposure, toxicity, and assay artifacts also matter.
Should EC50 and IC50 be log transformed?
Fitting is often parameterized on log concentration, and between-run summaries often use log values. The correct approach depends on the statistical objective; always report the final concentration and unit clearly.
Can IC50 be converted directly to Ki?
Not universally. Conversion depends on binding model, substrate or ligand concentration, affinity assumptions, and experimental design.