Insulin Oligomer Detection Protocol

Insulin samples (2 μL) were spotted onto a nitrocellulose membrane (0.22 μm, GE Healthcare Life Sciences, Buckinghamshire, UK). The membrane was blocked with 5% skim milk in 5% BSA/Tris-buffered saline (TBS) containing 0.01% Tween 20 for 1 h at room temperature. Subsequently, the membrane was incubated with primary anti-insulin antibody (1:3000, ab181547, Abcam, Cambridge, MA, USA) and anti-oligomer antibody (1:3000, A11, Invitrogen, Carlsbad, CA, USA) for 24 h. The following day, the membrane was incubated with the secondary anti-rabbit IgG antibody (1:10,000; R&D Systems, Minneapolis, MN, USA) for 1 h, as described^{10 (link)}. After washing with TBST, proteins were visualized using an ECL blotting detection kit (Bio-Rad) according to the manufacturer’s instructions. Luminescence was detected with a Las 4000 mini luminescent image analyzer (Fujifilm, Tokyo, Japan) using the Image Reader Las 4000 software. ImageJ was used to determine the intensity of each dot, which was used to quantify the amount of antibodies on the membrane.