Western Blot Protein Detection Protocol

Proteins were separated by electrophoresis on 8% or 10% 29:1 polyacrylamide gels in 1× TGS buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) and transferred onto nitrocellulose membranes using the Bio-Rad TransBlot system. Membranes were saturated by 30 min incubation in TBS-T buffer (Tris-buffered saline with 0.1% Tween 20), containing 5% (w/v) powder milk. Specific proteins were detected by incubation in TBS-T with the following antibodies: anti-HA-HRP (Clone 3F10, Roche) diluted 1000-fold; anti-Dbp6 polyclonal antibodies diluted 10 000-fold (36 (link)); anti-PGK1 (clone 22C5D8, Invitrogen) diluted 10 000-fold; rabbit peroxidase anti-peroxidase soluble complex (PAP, Sigma) diluted 10 000-fold. After incubation with primary antibodies, membranes were washed three times with TBS-T. Anti-rabbit or mouse IgG–HRP conjugate (Promega) were used, when needed, as secondary antibodies. After three washes of the membranes with TBS-T, proteins associated with antibodies were detected as chemiluminescent signals using Clarity Western ECL Substrate (Bio-Rad) and the ChemiDoc imager apparatus (Bio-Rad) followed by quantification with the ImageLab software (Bio-Rad).