Anti mouse horseradish peroxidase conjugated iggs

To assay protein expression levels via the ImageJ software for CAT-ELISA data normalization, 30 μg from each total cell lysate was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE). Following transfer, nitrocellulose membranes were blocked with 5% non-fat dry milk in PBS Tween 0.05% (PBS-T) for 1 h at room temperature. Mouse monoclonal primary antibodies specific to EGFP (Santa Cruz Biotechnology, Inc., B-2 clone, #sc-9996) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, Inc., G-9 clone, #sc-365062) were added for an additional incubation period of 1 h at room temperature. The membranes were washed in PBS-T and were incubated 1 h at room temperature with, as the secondary antibody, anti-mouse horseradish peroxidase-conjugated IgGs (#31430, Thermo Fisher, Waltham, MA, USA). All antibodies used were diluted in PBS-T containing 5% non-fat dry milk. Finally, the signal was detected by enhanced chemiluminescence with a Fusion FX7 apparatus (Vilber, Collégien, France).

For each sample, a total cell extract quantity of 50 μg was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred onto nitrocellulose membranes. The membranes were blocked in PBS-Tween 0.05% (PBS-T) containing 5% of nonfat dry milk for 1 h at room temperature prior to the addition of mouse monoclonal primary antibodies specific to EGFP for 1 h (Santa Cruz Biotechnologies, B-2 clone). The membranes were then washed three times with PBS-T and incubated for 1 h at room temperature with anti-mouse horseradish peroxidase-conjugated IgGs (Thermo Fisher, Waltham, MA) diluted in PBS-T and 5% nonfat dry milk. The signal was detected by enhanced chemiluminescence with a Fusion FX7 apparatus (Vilber, Collégien, France). The bands were then analyzed with the ImageJ software to determine the EGFP-Rev expression level for CAT-ELISA data normalization.