Hrp conjugated goat anti rabbit secondary antibody

Dot-blot assays were performed using the anti-amyloid fibril antibody LOC (Millipore, Burlington, MA, US, [86 (link)]), specific for amyloid fibrils. 20 µL of each sample containing Aβ(1-42) with or without Cu²⁺ or Cu⁺ was taken directly from the aggregation reactions and added to a 96-well dot-blot hybridization manifold (Scie-plas Ltd., Cambridge, UK). Vacuum pressure was applied to deposit the samples onto an LF-PVDF membrane (Trans-Blot, BIO-RAD, Hercules, CA, US). The membrane was dried and blocked with 30 mL of 5% skimmed milk in phosphate buffer saline with Tween-20 (PBS-T) and incubated for 1 h at room temperature. The membrane was then incubated for 30 min at room temperature with 30 mL of a primary antibody (1:100,000) dissolved in BSA/PBS-T. After three washes (5 min each time) with PBS-T, the membrane was incubated with a 1:20,000 dilution HRP-conjugated goat anti-rabbit secondary antibody (Sigma, St. Louis, MO, US). Thereafter, the membrane was washed three times with PBS-T (15 min × 1, 5 min × 2). The resulting dots were developed using an enhanced chemiluminescence solution (ECL, GE healthcare, Chicago, IL, US) together with the chemiluminescence detection system of a ChemiDoc^TM gel scanner (BIO-RAD, Hercules, CA, US). The densiometric analysis was performed using the instrument’s built-in software and the region-of-interest function.

Following electrophoresis on polyacrylamide gels, proteins were electro-transferred to nitrocellulose membrane (Trans-Blot Turbo Mini Nitrocellulose Transfer packs; Bio-Rad) using a Trans-Blot Turbo Transfer System (Bio-Rad). After protein transfer, the membranes were blocked for 1 h in 5% (w/v) milk in PBS at room temperature, and then immunoblotted in 5% milk and 0.1% Tween-20 in PBS with anti-insulin antibody produced in rabbit (SantaCruz, Dallas, TX, USA). Then, blots were incubated with HRP-conjugated goat anti-rabbit secondary antibody (Sigma-Aldrich), and immune reactive bands were detected using an enhanced chemiluminescence method (ECL kit; BioRad). Densitometry was analyzed by using the NIH Image (v1.63) software (National Institutes of Health, Bethesda, MD, USA). The pixel intensity for each region was calculated, the background was subtracted, and the protein expression results were normalized with respect to the bands of an anti-α-tubulin antibody (Abcam, Cambridge, UK) as loading control for each lane.

The CNS, FM and columellar muscle (CM) were homogenized in ice cold 50 mM Tris-HCl buffer containing 0.1% Triton X–100 and a protease inhibitor cocktail. After centrifugation at 12,000×g at 4°C for 20 min, the supernatant was collected and processed on an 8% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). The protein concentration of samples was determined by Bradford method and the average protein content of the samples loaded on the gel was the same. After electrophoresis, proteins were blotted onto PVDF Immobilon-P membrane (Millipore). Membranes were blocked with 5% non-fat milk at room temperature and thereafter incubated overnight at 4°C with anti-nAChR α7 (ab10096,1 µg/ml, Abcam, Cambridge, UK or ANC-007, 4 µg/ml, Alomone, Jerusalem, Israel) or anti-nAChR α4 (ANC-004, 4 µg/ml, Alomone, Jerusalem, Israel) primary antibody. In preadsorption controls the proportion of antigens and their immunogens were 1∶4 (ab10096), 1∶5 (ANC-007) and 1∶2 (ANC-004). After incubation with HRP-conjugated goat anti-rabbit secondary antibody (1∶10.000, Sigma, Budapest, Hungary), the primary labeled bands were visualized with ECL substrate (WesternBright, Advansta, Menlo Park, CA, USA or Pierce ECL Western Blotting Substrate, Rockford, IL, USA).