Xl 2000 sonicator

Human insulin dissolved in 25 mM HCl at a concentration of 2.0 mg/ml (344 μM in monomer units) was used as a fundamental sample solution. Fibrillation reaction was performed by incubating this solution at 65 °C for 24 h. For inducing polymorphism of amyloid fibrils, two types of additives, 100 mM NaCl or 100 µM SDS were used in addition to the solvent condition without additives. After the formation of amyloid fibrils, which were referred to as parent fibrils, a seed-dependent fibril formation was performed to obtain daughter fibrils. In this reaction, the fragments of fibrils were added to 2.0 mg/ml insulin dissolved in 25 mM HCl as seeds at a final concentration of 5% (w/v), and then incubated at 37 °C for 24 h. It should be noted that all of the seed-dependent fibrillation reactions were performed in the same solvent conditions without any additives. For the preparation of seeds, the parent fibrils were subjected to pulsed sonication using an XL2000 sonicator (Misonix, San Diego, CA, USA) operating for 1 s with power level at 2.0 W. The total number of sonication pulses was 40, and it was ensured that amyloid samples are not excessively heated during the sonication treatment.

IP was performed with Dynabeads (Thermo Fisher Scientific) according to the manufacturer’s protocol. The preincubation of Dynabeads (100 µl) (M-280 Sheep Anti-Mouse IgG or M-280 Sheep Anti-Rabbit IgG) with primary antibody (5.0 µl) in 1.0% BSA/PBS was performed at 4°C overnight. The beads conjugated with primary antibodies were then washed with IP buffer (10 mM Tris–HCl [pH 7.8] [Nacalai Tesque], 1.0% NP-40 [Nacalai Tesque], and 15 mM NaCl [Nacalai Tesque]/EDTA-free protease inhibitors [100×] [Nacalai Tesque]). Cells were collected with a cell scraper in IP buffer. A total of 1.0 × 10⁷ cells were sonicated on ice three times for 1 s by the XL-2000 sonicator (MISONIX). Sonicated cells were centrifuged at 21,900g for 10 min. The supernatant was collected and incubated with primary antibody conjugated with Dynabeads at 4°C overnight. After incubation, the sample tubes were set on DynaMag-2 (Thermo Fisher Scientific), and Dynabeads were washed three times with IP buffer. Dynabeads were then suspended with fresh IP buffer and incubated at 95°C for 5 min. The supernatant was collected on DynaMag-2 and mixed with 2 × Laemmli sample buffer. Immunoblotting was performed as described.