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. 2021 Jan 6;120(3):476–488. doi: 10.1016/j.bpj.2020.12.019

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Characterization of TMR-IAPP/Atto425-DnaK complexes using TCCD. (A) SEC profile measured by absorbance at 280 nm of a solution containing 4 μM TMR-IAPP and 2 μM atto425-DnaK is shown. The fraction eluted at 17 min is examined using TCCD. (B) Photon counts/ms obtained from atto425-DnaK (lower panel) and TMR-IAPP (upper panel) using excitation with the blue (λ = 450 nm) laser are shown. (C) Histogram of the FRET efficiency (ε = I_TMR/[I_atto425 + I_TMR]) of the coincident bursts is shown. (D) Dependence of ε on the total burst intensity is shown. (E and F) Histograms of the apparent number of monomers (E) and approximate stoichiometry (F) of TMR-IAPP and atto425-DnaK molecules in the complexes are shown. Estimates in (F) are obtained using the coincident bursts with both (λ = 450 and 543 nm) lasers on. To see this figure in color, go online.