Gaining insight into optimal experimental design. The approximate posterior probability distributions for Kd (A) have different shapes if the data analyzed was simulated from three cells containing equal concentrations of donors and acceptors which are much lower than (left), higher than (right) or about equal to Kd (centre). For the data analyzed in the left panel, for instance, the cells contained the concentrations [D0] = [A0] = 0.2·10-3 μM, [D0] = [A0] = 1·10-3 μM, and [D0] = [A0] = 5·10-3 μM. Insets show amount of complex formed as a function of Kd for the indicated concentrations, demonstrating that where complex formation is insensitive to Kd corresponds to plateaus in the posterior probability distributions for Kd. In each plot (or inset), a vertical dashed line (or red circle) indicates 10-6M, the true value of Kd. (A) had 36,000 steps/walk and 5% added noise. Exploring another aspect of fluorophore concentrations, increasing the ratio [D0] : [A0] increases the uncertainty in fitting Kd (B). As the ratio was increased (by keeping [D0] constant for the three cells at 0.2·10-6M, 1.0·10-6M and 5.0 10-6M while decreasing [A0] according to the ratio), posterior probability distributions for Kd broadened (true value indicated by dashed vertical line). Insets show data used for fitting (bars marked 'Efr = 0.4') from the donor channel (left) and FRET channel (right) contrasted with data from the same cells simulated with Efr = 0, demonstrating that the relative contribution of FRET decreases as [D0] : [A0] increases. (B) had 50 measurements/cell/channel, 36,000 steps/walk and 3% added noise. Bars show mean ± SD. For other parameters, see Methods.
Lichten and Swain BMC Biophysics 2011 4:10 doi:10.1186/2046-1682-4-10