Trans blot wet transfer unit

Briefly, 50 μg of protein extract of LV from four individual animals of each group (HRP and LRP) were resolved on 12.5% SDS-PAGE and transferred onto nitrocellulose membranes (Hybond, UK), using a trans-blot wet transfer unit (Bio-Rad) by applying a current of 100 mA at 4°C overnight. Equal total protein loads were confirmed by Ponceau red (0.1% Ponceau red, 5% acetic acid, v/v) staining of the nitrocellulose membranes. The membranes were rinsed with TBS-Tween 0.1% and incubated with blocking buffer (5% low-fat milk powder in TBS-Tween 0.1%) at room temperature for 2 hours. Trans-blotted proteins were then probed overnight at 4°C with a rabbit polyclonal anti-desmin antibody (Santa Cruz, USA) at dilutions of 1:500. After washing three times in TBS-Tween 0.1% for 5 min, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilution) in blocking solution for 2 h.
Specific binding was revealed with a western blotting detection ECL system (Amersham, UK) and exposed to a CCD camera (ChemiDoc, Bio-Rad). Desmin signals were processed by Image Lab Software (Bio-Rad) and the signal intensities were measured in delimitated areas of equal size and reported as arbitrary units.

Briefly, 60 μg of protein extract of PFC from four animals of each group were resolved in a denaturing SDS polyacrylamide gel – 15% (Laemmli, 1970 (link)) and colorburst^TM electrophoresis markers (Sigma^®) were used as molecular mass standards. The proteins were transferred onto nitrocellulose membranes (Hybond, Amersham, United Kingdom), using a trans-blot wet transfer unit (Bio-Rad^®), by applying a current of 100 V at 4°C overnight.
Next, the membranes were rinsed with TBS-Tween 0.1% and incubated with blocking buffer (5% low-fat milk powder in TBS-Tween 0.1%) at room temperature for 2 h. Trans-blotted proteins were then probed overnight at 4°C with a polyclonal anti-primary antibody directed against BDNF [1:500 (Alomone^®)] and a polyclonal anti-GAPDH [1:2000 (Santa Cruz)]. After washing three times in TBS-Tween 0.1% for 10 min, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1: 2,500 dilution) in blocking solution for 1 h.
Specific binding was revealed with a Western blotting detection ECL system (Amersham, United Kingdom) and exposed to a CCD camera (ChemiDoc, Bio-Rad^®). ProBDNF, mBDNF, and GAPDH signals were processed by Image Lab Software (Bio-Rad^®) and the signal intensities were measured in delimitated areas of equal size and expressed as arbitrary units. Protein levels of GAPDH were used to normalize the results.