Monitoring membrane binding and insertion of peptides by two-color fluorescent label

►We developed an approach for monitoring membrane binding and insertion of peptides using a fluorescent environment-sensitive label of the 3-hydroxyflavone family. ►The dual emission of the label covalently bound to the N-terminus of three peptides correlates well with the depth of its insertion measured by the parallax quenching method. ►Orientation of the label bound to the peptides can also be determined by two photon microscopy on giant vesicles.