Bl ac10ds a2 cantilever

Real-time monitoring of amylin aggregation was performed using a HS-AFM instrument operated on tapping mode equipped with an Olympus BL- AC10DS-A2 cantilever of spring constant k=0.1 Nm⁻¹ and frequency f=~400 kHz in water. The instrument setup as described in a recently reported study.[12 (link)] Freshly dissolved amylin monomers (5 μM) dissolved in 30 mM NaAc buffer were incubated for ~2–3 days at 37 °C under continuous agitation to form amyloid fibers. The amyloid seeds were generated by sonicating the fibers with a handheld sonicator and verified using AFM imaging (UR-21P, TOMY). The copolymer solutions of 11μg/mL were prepared in 30 mM NaAc buffer, pH 5.5. The fibril seeds were incubated on mica surface for ~5 minutes, rinsed with buffer to remove the unbound seeds following immersion in 60 μL of buffer. The fibril seeds on mica surface were first confirmed using imaging and the 60 μL of buffer was replaced with 5 μM of freshly dissolved amylin monomers (fibril seeds:monomer=1:19 (v/v)) in the absence and presence of 11μg/mL of SMAEA or SMAQA following HS-AFM imaging. The HS-AFM videos were analyzed using ImageJ (NIH).