FIGURE 2.

FRET is sensitive to subunit labeling. A, binding curves for Cdc11 AF555 and Cdc11 AF647 using varying concentrations of Cdc11^AF555 (25 nm Cdc11^AF555 (red), 35 nm Cdc11^AF555 (cyan), 50 nm Cdc11^AF555 (magenta)). B, schematic showing labeling of septin octamer at different subunits. The Cdc11^AF555 is kept constant, whereas the acceptor (AF647) is transferred along the septin octamer. This schematic corresponds to the results in C and D. C, Coomassie gels and typhoon scans of labeled septin octamers with AF555 and AF647. D, the red curve shows the predicted efficiency of transfer from the FRET equation. The black circles with S.E. error bars correspond to experimental data for a 1:4 mixture of Cdc11^AF555:acceptor-labeled septin. The data points are labeled for the labeled septins (donor-acceptor) in the FRET system.