Which EC50?

The experiment depicted below (from John Herich, who suggested I write this article) measures cAMP production as a function of increased agonist concentration. The assay used to measure cAMP is based on measuring fluorescence. Higher concentrations of cAMP in the sample compete for fluorescent labeled cAMP for binding to an antibody, which is measured.

The red data and curve show the change in fluorescence, normalized to percent . The EC₅₀ is about 11nM. Each fluorescent value was then transformed to the corresponding concentration of cAMP (by interpolating from a standard curve). The blue data and curve show the cAMP accumulation, normalized to percentages. The EC₅₀ is 178 nM -- 16.7 times higher than the other EC₅₀. The EC₅₀ of cAMP production tells us about the biology of the system, while the EC₅₀ for changes in fluorescence really tells us nothing useful.

Why the difference? The relationship between fluorescent signal and cAMP production is not linear, so the EC₅₀ for fluorescence and the EC₅₀ for cAMP production are very different. When you think about the meaning of an EC₅₀ value, always think about which response the EC50 is computed for.

The same principle applies, of course, inside a cell. Depending on which second messenger or response your assay, you'll get different EC₅₀ values.