Semi dry electroblotting transfer unit

Protein samples were suspended in cracking buffer (2% SDS, 10% Glycerol, 60 mM Tris-Cl pH 6.8, 0.01% Bromophenol Blue, and 100 mM DTT) and incubated for 5 min at 100°C. Protein electrophoresis was performed at 120 V on a 12% SDS-PAGE gel. Gels were stained in Coomassie-Blue solution (20% methanol, 10% acetic acid).
For Western Blot analysis, proteins were transferred to a nitrocellulose membrane (Immobilon – Merck Millipore Ltd) for 55 min at 15 V using a semi-dry electroblotting transfer unit (Bio-Rad, Hercules, CA, USA). Membranes were incubated for 1 h with blocking buffer (1% dry skim milk, 0.1% Tween in PBS) and then incubated for 1 h with primary antibody diluted in blocking buffer (1/500). After washing with PBS-0.1%Tween, membranes were incubated for 1 h with secondary antibody labeled with IRDye fluorophores (LI-COR, Lincoln, NE, United States) diluted in blocking buffer (1/20,000). Finally, the membranes were scanned using the Odyssey Imaging System (LI-COR).
Proteins were quantified with the UV–Vis NanoDrop One spectrometer (Thermo Scientific).

Protein samples were treated with cracking buffer (50 mM Tris-HCl pH 6.8, 2% wt/vol sodium dodecyl sulfate, 0.1% wt/vol bromophenol blue, 10% wt/vol glycerol, and 50 mM dithiothreitol) and incubated at 100°C for 5 min. Protein electrophoresis separation was performed at 120 V for 1 h on a 10% SDS-PAGE gel. Subsequently, proteins were transferred to a nitrocellulose membrane using a semi-dry electroblotting transfer unit (Bio-Rad, Hercules, CA, United States) for 45 min at 15 V. After 1 h blocking buffer [0.1% Tween 20 and 1% dry skim milk in phosphate-buffered saline (PBS) buffer], membranes were incubated for 1 h with the corresponding primary antibody. Membranes were washed four times during 5 min with washing buffer (0.1% Tween 20 in PBS buffer) and incubated for 1 h with a 1:20.000 blocking buffer dilution of IRDye fluorophore-labeled secondary antibodies (LI-COR, Lincoln, NE, United States). After washing four times, membranes were scanned using the Odyssey Imaging System (LI-COR).

Protein samples were dissolved in a cracking buffer and incubated for 5 min at 100°C. Protein electrophoresis was performed at 120 V on 12% SDS-PAGE gel. Gels were stained in a Coomassie Blue solution (20% methanol and 10% acetic acid).
For Western blot analysis, proteins were transferred to a nitrocellulose membrane for 55 min at 15 V using a semi-dry electroblotting transfer unit (Bio-Rad, Hercules, CA, the United States). Membranes were incubated for 1 h with blocking buffer (1% dry skim milk and 0.1% Tween in PBS). Then, membranes were incubated for 1 h with primary antibody diluted in blocking buffer (1/500). After washing with PBS-0.1% Tween, membranes were incubated for 1 h with IRDye fluorophore-labeled secondary antibodies (LI-COR, Lincoln, NE, the United States) diluted in a blocking buffer (1/20,000). Finally, membranes were scanned using the Odyssey Imaging System (LI-COR).