Image station 4000mm digital imaging system

After cell lysis, protein concentration was measured by Bradford method. Twenty-five μg of denatured total protein were loaded onto a 10% polyacrylamide gel and were run for 30 min at 200 V. Proteins were transferred to PVDF membrane (Millipore, Billerica, MA, USA) for 1.5 h at 100 V. Ponceau staining was applied to determine blotting efficacy. Membranes were blocked with 3% w/v non-fat dry milk (Bio-Rad) in TBS for 1 h followed by incubation with the primary antibodies at 4°C overnight. After the washing steps (0.05 v/v% Tween-20 in TBS), appropriate secondary antibody was applied for 1 h, signals were detected by SuperSignal West Pico Chemiluminescent Substrate Kit (Pierce/Thermo Scientific, Part No.: 34080), and visualized by Kodak Image Station 4000MM Digital Imaging System. WB analyses were repeated 3 times. Ponceau staining was used to assess the equal loading of samples. Applied antibodies are indicated in S4 Table.

For caseinase and gelatinase zymogram analysis, 20 μL CCM was used, and normal human serum as control [19 (link),25 (link)]. Protease activities were visualized by Coomassie Blue staining (Bio-Rad Laboratories GmbH). Densitometry was carried out on a Kodak Image Station 4000MM Digital Imaging System using Kodak Molecular Imaging Software v. 4.0.3 (Eastman Kodak Company, Rochester, NY, USA).

Sera collected before (3 wpi) and after (6 wpi) the induction of the anti-SAV E2 antibody response were analyzed to determine the total IgM content by western blot. Total serum protein was quantified using a Micro BCA assay (Thermo Fisher Scientific) and 0.5 μg was loaded onto 4–12% gradient NuPAGE Novex Bis-Tris gels after denaturation with a 5 μL LDS buffer (4×) at 70°C for 10 min. Samples were subjected to SDS-polyacrylamide gel electrophoresis with a 1× MOPS buffer for 50 min at 200 V and 120 mA (Invitrogen). MagicMark^TM XP and SeeBlue Plus 2 pre-stained were used for molecular weight estimation (Invitrogen). Protein was blotted onto a polyvinylidene difluoride (PVDF) membrane, blocked and incubated overnight with anti-trout IgM mAb (IgF1-18 (6-1-18); 1:200). After four washes, the membrane was incubated with HRP conjugated anti-mouse (1:5000 dilution; Santa Cruz Biotechnology) for 1 h and developed using SuperSignal West Femto Trial Kit (Thermo Fisher Scientific) and a KODAK Image Station 4000MM Digital Imaging System. Band intensities (arbitrary unit, AU) were determined by subtracting the background signal from the visualized IgM band of ∼70 kDa.

The eluates from the IPs/co-IPs were directly subjected to SDS–PAGE using NuPAGE Novex Bis-Tris 4–12% gels (Life Technologies) and then proteins were transferred to PVDF membrane and blocked for one h in 5% fat free dry milk or 5% BSA in TBS-T and then incubated with the primary antibody at 4 °C, then the membranes were treated with the secondary antibody for 1 h.
In this study two different imaging systems were used; the first one was KODAK Image Station 4000MM Digital Imaging System where membranes were blocked with dry milk or BSA, secondary antibodies were HRP conjugated and the blots were developed using SuperSignal West Pico Chemiluminesccent Substrate (Pierce). The second system was Odyssey CLx infrared imaging system where specific blocking buffer from LI-COR was used and the secondary antibodies were infrared dye conjugated. The membranes were stripped by incubating in 0.2 M NaOH in 15 min and process was repeated for the primary antibody specific for the precipitated factor.