Owl hep 1 semidry electroblotting system

Tris-tricine SDS-PAGE was performed as previously described (46 (link), 47 (link), 48 , 49 (link)). Samples for electrophoresis were combined with SDS-PAGE loading dye (50 mM Tris-Cl [pH 8.45], 100 mM dithiothreitol, 2% [w/v] SDS, 10% [v/v] glycerol, 0.1% [w/v] bromophenol blue). Electrophoresis was performed in the presence of Tris-tricine SDS-PAGE cathode buffer (0.1 M Tris, 0.1 M Tricine [pH 8.45], 0.1% [w/v] SDS) and anode buffer (0.2 M Tris-Cl [pH 8.9]).
Western transfers were performed using the Owl HEP-1 Semidry Electroblotting System (ThermoFisher Scientific) semi-dry transfer method. Following electrophoresis, SDS-PAGE gels were transferred onto a polyvinylidene fluoride membrane (Millipore). polyvinylidene fluoride membranes (whole or in strips) were subjected to immunoblot analysis with specific primary antibodies and secondary antibodies (anti-mouse/rabbit IgG coupled to horseradish peroxidase; Sigma Aldrich; 1:5000 dilution in blocking buffer). Detection of chemi-luminescent signal was performed with Clarity ECL Western Blotting substrate (BioRad) and imaged using a ChemiDoc MP Imaging system (BioRad). Immunoblot quantitation was performed on three independent biological replicates using Image Lab software (BioRad).

To study clusterin N-glycosylation patterns, as well as to monitor clusterin proteoforms in the ERF and blood plasma, 2D western blot analysis was performed. Pooled samples of both the ERF and plasma were prepared for each subgroup. All samples were normalized to the same protein amount. Following SDS-PAGE (10% resolving gel), the proteins were transferred to a PVDF membrane (10V constant voltage for 60 min) using an Owl HEP-1 semi-dry electroblotting system (Thermo Scientific, Waltham, MA, USA). After incubation with a blocking buffer (3% BSA in PBS) at 30°C for 60 min, the membranes were first incubated with primary antibody (CL7757 AP; Cedarlane, Burlington, Ontario, Canada) (1:10,000 dilution) and, then, with secondary goat anti-rabbit IgG (whole molecule) antibody conjugated with peroxidase (1:5,000 dilution) (A6154; Sigma-Aldrich, Prague, Czech Republic). Visualization was performed using a chemiluminescent substrate (SuperSignal West Pico; Thermo Scientific, Waltham, MA, USA) and CL-XPosure film (Thermo Scientific, Waltham, MA, USA). The 2D western blots were digitized (1200 dpi, 16-bit grayscale) and processed by Progenesis SameSpots software (Nonlinear Dynamics, Newcastle upon Tyne, UK). The expression profiles were based on spot normalized volumes.