Triton X‐100 fluorescence

Triton X‐100 has an excitation and emission wavelength of 275 and 302 nm, respectively. ShHTL7, ShHTL7‐L143Y, AtD14, and OsD14 were incubated in 50 mM Tris–HCl pH 7.5, 150 mM NaCl, with or without 0.005% Tween‐20, at concentrations ranging from 10 μM to 1 nM (obtained using serial dilutions) with 154 nM of Triton X‐100 for 1–2 h, and fluorescence values were then measured by SpectraMax i3 (Molecular Devices) at excitation and detection wavelengths of 275 and 302 nm, respectively. The fluorescence exhibited by Triton X‐100 alone was subtracted from the fluorescence in the presence of protein. Data were normalized to the background fluorescence from the protein to obtain the change in Triton X‐100 fluorescence intensity. To determine the K _d, the changes in Triton X‐100 fluorescence intensity were plotted against the concentration of the protein using the single binding site model implemented in GraphPad (Prism 6).