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. 2014 Jul 17;9(7):e102572. doi: 10.1371/journal.pone.0102572

Figure 6. Determination of A/D ratio for optimal FRET between Alexa Fluor 488-LFA-1 and Alexa Fluor 555-D1-D2-Fc.

Figure 6

Fluorescence emission scans were obtained for the Alexa Fluor 488-LFA-1 and Alexa Fluor 555-D1-D2-Fc mixture, where the concentration of Alexa Fluor 555-D1-D2-Fc was kept constant at 100 nM and that of Alexa Fluor 488-LFA-1 was varied: 25 nM (A/D = 8.40), 50 nM (A/D = 4.20), 100 nM (A/D = 2.12), 150 nM (A/D = 1.41), 200 nM (A/D = 1.06), 250 nM (A/D = 0.84) and 300 nM (A/D = 0.70). The mixtures were excited at 470 nm. The highest FRET efficiency (∼53.51%) was obtained for A/D = 2.12. For both 4.20 and 8.40 A/D ratios, the donor peak intensity is very small compared to the acceptor peak, while for 2.12 A/D ratio, the donor peak intensity is higher but not overwhelming the acceptor peak intensity. These higher emission counts at 4.20 and 8.40 A/D can be attributed to the direct emission of the acceptor as the acceptor concentration exceeds the saturating concentration required to saturate the donor binding pairs.