Monoclonal antibody directed against the flag epitope

Whole-cell protein extracts were prepared in Laemmli buffer³⁶ (glycerol 5% v/v, β-mercaptoethanol 2.5%, SDS 1.15% p/v, Tris–HCl 31 mM pH 6.6 and bromophenol blue 0.05%). Protein samples were analyzed by Tris-Glicine-SDS triphasic gels with 16.5% polyacrylamide. Proteins were transferred from the gels to PVDF membranes by using the semidry electrophoretic transfer cell (Bio-Rad) at 15 V during 40 min. For the Western blot analysis, a monoclonal antibody directed against the Flag-epitope (Sigma-Aldrich) diluted 1:10.000 in a solution of PBS, 0.2% Triton and 3% skimmed milk was used. The membranes containing the proteins were incubated with the diluted antibody for 16 h at 4 °C. The membranes were afterwards washed for 10 min with PBS, 0.2% Triton X-100 (Sigma-Aldrich) solution. The washing step was repeated three times. Thereafter, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Promega) diluted 1:2.500 in a solution of PBS, 0.2% Triton X-100 for 45 min at room temperature. Again, three washing steps of 30 min with PBS, 0.2% Triton solution were performed, and immunodetection of the specific protein was performed by enhanced chemiluminescence by using the Molecular Imager ChemiDoc XRS system and Quantity One software (Bio-Rad).

Whole-cell protein extracts were prepared in Laemmli buffer (glycerol 5% vol/vol, β-mercaptoethanol 2.5%, SDS 1.15% p/v, Tris–HCl 31 mM pH 6.6 and bromophenol blue 0.05%). Protein samples were analyzed by Tris-Glycine-SDS triphasic gels with 16.5% polyacrylamide. Proteins were transferred from the gels to PVDF membranes by using a semidry electrophoretic transfer cell (Bio–Rad) at 15 V for 40 min. For Western blot analysis, a monoclonal antibody directed against the Flag epitope (Sigma–Aldrich) or a polyclonal antibody directed against the IrmA protein were diluted 1:10.000 or 1:1000 in a solution of PBS, 0.2% Triton and 3% skimmed milk. The membranes containing the proteins were incubated with the diluted antibody for 16 h at 4°C. The membranes were then washed for 10 min with PBS and 0.2% Triton X-100 (Sigma–Aldrich) solution. The washing step was repeated three times. Thereafter, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Promega) or anti-rabbit IgG (Promega) diluted 1:2.500 in a solution of PBS and 0.2% Triton X-100 for 45 min at room temperature. Again, three washing steps with PBS and 0.2% Triton solution were performed for 30 min each, and immunodetection of the specific protein was performed by enhanced chemiluminescence by using the Molecular Imager ChemiDoc XRS system and Quantity One software (Bio–Rad).

Protein extracts were prepared in Laemmli buffer⁷⁵ (5% glycerol, 2.5% β-mercaptoethanol, 1.15% SDS, Tris-HCl 31 mM pH 6.6 and 0.05% bromophenol blue). Protein samples were analyzed in 16.5% polyacrylamide Tris-tricine-SDS triphasic gels. After electrophoresis, the proteins were transferred from the gels to PVDF membranes using a semidry electrophoretic transfer cell (Bio-Rad) at 15 V for 40 min. For the Western blot analysis, a monoclonal antibody directed against the Flag epitope (Sigma-Aldrich) diluted 1:10,000 in a solution of PBS, 0.2% Triton and 3% skim milk was used. The membranes containing the proteins were incubated overnight with the diluted antibody at 4 °C. After incubation, the membranes were washed three times for 10 min with a solution of PBS and 0.2% Triton X-100 (Sigma-Aldrich). Thereafter, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Promega) diluted 1:2500 in a solution of PBS and 0.2% Triton X-100 for 45 min at room temperature. After incubation, three additional washing steps with PBS and 0.2% Triton solution were performed for 30 min each time. The immunodetection of the specific protein was performed by enhanced chemiluminescence using a Molecular Imager ChemiDoc XRS system and Quantity One software (Bio-Rad).