Quantitative Dot Blot Protein Analysis

Proteins (3 μg) were directly loaded onto nitrocellulose Trans-Blot transfer 0.45 μm (Bio-Rad) membranes, depositing each sample on the membrane by vacuum filtration on a Bio-Dot Microfiltration Apparatus (Bio-Rad), as described previously [41 (link)]. Dot blot membranes were blocked with 3% (w/v) BSA (Sigma) and 0.1% (v/v) Tween 20 in Tris-buffered saline, pH 7.5, incubated with mouse monoclonal anti-NT (1:1000; Merck-Millipore), then with anti-mouse peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology Inc.). Blots were developed with Luminata™ Forte Western Chemiluminescent HRP Substrate (Millipore) on the ChemiDoc XRS system (Bio-Rad). Densitometry was done with Progenesis PG240 v2006 software (Nonlinear Dynamics). NT immunoreactivity was normalized to the actual amount of proteins loaded on each dot in the membrane as detected after Ponceau Red staining (Fluka). Values were expressed as mean ± SEM.