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).
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).