Figure 4. FRET-based assay (A) and EIA-based assay (B) for PE/PC binding of ankBDn and its mutants.

A) Differences in binding affinity of ankBDn and its mutant shown by FRET-based assay. FRET between TRITC-labeled proteins and PE/PC liposomes containing NBD-PE was detected as a decrease in NBD-lipid fluorescence emission intensity upon protein binding to the liposomes. As a control of nonspecific binding, TRITC-labeled IgG was used. Average values were calculated from at least three independent experiments. Error bars indicate standard deviation. B): Binding of recombinant proteins by PE/PC liposomes. EIA assay: a multi-well plate was coated with PE/PC liposomes and incubated with the indicated proteins. Anti-His-Tag antibodies were used for protein detection and anti-mouse-STAG antibodies were used for electrochemiluminescence reaction. Thermally denatured ankBDn was used as a negative control. Average values calculated from at least three independent experiments. Error bars indicate standard deviation.