Dot blot analysis

Nitrocellulose (Hybond ECL, GE Healthcare, Chicago, IL) was pre-wetted with PBS-T (PBS containing 0.1% Tween 20) and then spotted under vacuum with 50 µl 10% seminal plasma, 10% blood plasma, or 20 µg/ml semen amyloids (SEVI or SEM1(86–107) fibrils) or the corresponding monomeric peptides. Wells were then washed once with PBS-T under vacuum. The nitrocellulose membrane was then immediately transferred to a blocking solution (PBS-T containing 5% non-fat dry milk and 1% BSA) and incubated at 4°C with gentle shaking for 2 hr. The membrane was then washed three times for 5 min before incubation with anti-Amyloid Fibrils OC Antibody (EMD Millipore, Billerica, MA) or Anti-Amyloid Oligomers A11 antibody (Abcam, Cambridge, United Kingdom) (both used at 1:2500 in PBS-T with 1% BSA). Primary antibody incubation was allowed to proceed overnight at 4°C with gentle shaking. The membrane was then washed three times for 5 min each before addition of HRP-conjugated rabbit IgG (GE Healthcare) (used at 1:5000 in PBS-T with 1% BSA). Secondary antibody incubation was allowed to proceed for 2 hr at 4°C with gentle shaking. The membrane was then washed three times for 10 min each, and then developed by 1 min incubation with the Western Lightning ECL Pro (Perkin Elmer, Waltham, MA), and exposed using chemiluminescence film (Amersham Hyperfilm ECL, GE Healthcare).