Hiapp

To prepare fibrillar hIAPP for binding studies, 30 μmol/l hIAPP (#60804, Anaspec, Fremont, CA, USA) was dissolved in 10 mmol/l phosphate buffer and 5 mmol/l NaCl and incubated overnight at room temperature with agitation. For saturation binding assays, concentrations of synthetic hIAPP fibrils and radiolabelled florbetapir were determined empirically starting with concentration ranges based on the literature and the authors’ experience with amyloid β (Aβ) peptide [32 (link)]. Concentration dependent binding assays were carried out using 0.5, 0.25, 0.125, 0.0625, 0.03125, or 0.015 μg of synthetic hIAPP fibrils and 0.15 nmol/l ¹⁸F-florbetapir in 250 μl PBS, eliciting binding of between 0 and 20% of the radioligand. Reaction mixtures contained IAPP fibrils (0.2 μg; probe sonicated) and ¹⁸F-florbetapir (0.9 nmol/l) in 250 μl PBS. Nonspecific binding was defined as the binding of the radioligand in the presence of 10 μmol/l BTA-1 [2-(4´-methylaminophenyl)benzothiazole] [33 (link)]. After incubation for 1.5 h at 37°C, bound radioactivity was harvested by vacuum filtration onto Millipore MultiScreenHTS 96-well glass fiber FB filter plates using a Millipore MultiScreenHTS Vacuum Manifold (Millipore, Billerica, MA, USA). Filters containing bound ¹⁸F-florbetapir were assayed for radioactivity in a gamma counter.

hIAPP and rIAPP (97% purity) was purchased from Anaspec (Fremont, CA). 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine sodium salt (POPS) were purchased from Avanti Polar lipids Inc. (Alabaster, AL). 6-carboxyfluorescein was purchased from Sigma-Aldrich (St.Louis, MO). Lucigenin was purchased from Invitrogen (Eugene, OR).