Figure 2.
Example of binding measurement using FCS performed under conditions of the “infinite dilution regime” satisfied at extremely high lipid excess over fluorescently labeled protein (see text). Acidification results in a progressive shift of mobility from that of the free T-domain to that of a vesicle-bound T-domain. Quantitative determination of membrane-competent fraction, FMC, is achieved by a linked analysis (solid lines) of fluorescence autocorrelation curves (dashed lines) that links the two correlation times for all curves and allows free fitting of the pre-exponential amplitudes (see Eq. 2-4, and (16)). For the purpose of better visual representation all data are normalized to the same number of fluorescent particles in the focal volume (Note that the absolute values of amplitudes are not important under these conditions, but only their relative contributions (Eq. 5).