*5.4. Tryptophan Fluorescence Quenching Assay for Ligand Binding to Purified, Reconstituted P-gp*

Binding of Aβ peptides to purified, reconstituted P-gp was measured by quenching of the intrinsic fluorescence of endogenous tryptophan residues as described [23] and modified [19]. However, imidazole was removed by ultra-centrifugation (70,000 g, 20 min 4 ◦C) of reconstituted P-gp and subsequent resuspension in binding buffer (20 mM MOPS, pH 8.0, 200 mM NaCl). P-gp (10–15 µg) was added to quartz silica cuvettes in a total volume of 300 µL. A tryptophan fluorescence emission spectrum was measured from 300–400 nm (emission slit width 5 nm) using excitation at 295 ± 10 nm at a scan speed of 120 nm/min. Lyophilised Aβ<sup>40</sup> and Aβ<sup>42</sup> peptides were resuspended in 2 mM NaOH at pH~10.5 to concentrations of 1.6 mg/mL (385 mM) and 1.0 mg/mL (221 mM) as described [89]. The peptides were added to sample cuvettes at concentrations in the range of 1–25 µM (Aβ42) or 1–50 µM (Aβ40). Sample cuvettes were held at a temperature of 37 ◦C and incubated for 20 min prior to measuring the fluorescence emission spectrum. Nicardipine (5 µM) was added following the final Aβ peptide addition to determine the maximal

possible quenching. The spectra obtained in the presence of nicardipine were subtracted from those containing Aβ peptide to remove background signal.
