Figure 2. Experimental validation for FRET-based stoichiometry assay.

(a) Bars depict average 3³-FRET (black) and E-FRET (red) efficiencies for various ECFP-EYFP concatemers as described in cartoon below (mean±s.e.m.; n, number of cells, as labelled for each bar). Note that the average 3³-FRET and E-FRET efficiencies are equal for ECFP-EYFP dimers but different for multimers. (b) The experimentally determined stoichiometry ratio, ν=E_A,max/E_D,max for each concatemer (grey symbol; mean±s.e.m.) follows the identity relation (black fit) with expected number of donor and acceptor molecules in the complex (n_D/n_A) confirming the theoretical relation equation 3.