4.5. Fluorescence Polarization Assay

For fluorescence polarization measurements, peptides were dissolved in DMSO at 10 mM and diluted in 10 mM HEPES (pH 7.5) containing 0.1% Tween-20. The assay was conducted on untreated black 384-well microtiter plates (low volume, non-binding surface, round bottom, non-sterile black polystyrene, Corning B.V. Life Sciences, Nr. 3676, Kennebunk, ME, USA) in a final volume of 10 µL, using 10 mM HEPES (pH 7.5) with 0.1% Tween-20 as a buffer. The final DMSO proportion in the assay was less than 0.1%. The measurements were performed on a SAFIRE II (Tecan) microplate reader, using an excitation wavelength of 470 nm and emission wavelength of 525 nm. The final assay mixtures contained 5 µL fluorescein-labeled peptide (furnishing a final assay concentration of 10 nM), and 5 µL of increasing amounts of Epsilon protein (final concentrations ranging from 0.05 nM to 5 µM). The negative controls comprised 5 µL fluorescein-labeled peptide (maintaining a final assay concentration of 10 nM) and 5 µL 10 mM HEPES buffer (pH 7.5) containing 0.1% Tween-20. To avoid protein aggregation, Tween-20 (0.1% final concentration) was used as a detergent in all experiments. After adding the fluorescent peptide, the plates were centrifuged (2000 min⁻¹, 3 min) and shaken briefly (2000 rpm for 10 min at room temperature), and polarization of the emitted fluorescence was directly recorded. In every case, the well containing only the fluorescein-labeled peptide was subtracted from the assay well as background. The polarization values (mP) were plotted as a function of the logarithm of the protein concentration. Binding curves were fitted by using GraphPad Prism software (version 4 for Windows, GraphPad, San Diego, CA, USA) employing a sigmoidal dose-response model (4 PL), and K_D values in the nanomolar range were obtained. All of the experiments were carried out in duplicate in three independent experiments, and the results are expressed as the mean ± standard deviation (SD).