I assume you are asking about fluorescence recovery after photobleaching. You must first define the measurement that is being inhibited (recovery time, as defined by a specific level of fluorescence), and you need to be able to measure the value for no inhibition (MAX) and 100% inhibition (MIN). In order to calculate IC50 you must first calculate % inhibition.
% inhibition = 100 x (1- (X-MIN)/(MAX-MIN)), where X is the measurement made at a given inhibitor concentration.
Although I have no personal experience with FRAP, I imagine that a problem with measuring % inhibition is that a fully inhibited result is that recovery never occurs, so there is no value for MIN.
An alternative approach would be to define an effective concentration (EC) that increases recovery time by a certain factor. For example, you might define the EC3 as the concentration of inhibitor that increases recovery time by 3-fold. The choice of the factor might depend on how reproducible the measurement of recovery time is. You want to be sure that you can make the distinction between a change in recovery time and experimental variability.
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