Mouse anti his antibody

Following electrophoresis, the proteins were transferred to a nitrocellulose membrane (110 V, 1 h, 4°C) using the Mini Trans-Blot cell apparatus (Bio-Rad). Nonspecific binding sites were blocked using PBS-Tween 20 (0.05% Tween 20, J.T. Baker) containing 5% non-fat dry milk for 1 h at room temperature. The membrane was then overlaid with mouse anti-His antibody (Bio-Rad), diluted 1:1000 in the blocking solution and incubated overnight at 4°C. Next day, the membrane was washed three times with PBS-Tween 20 and overlaid with peroxidase-conjugated goat anti-mouse antibodies (Jackson ImmunoResearch Laboratories) diluted 1:5000 in blocking solution, for 1 h at room temperature. The membrane was then washed three times with PBS-Tween 20. Before imaging (using the ChemiDoc MP imaging system, Bio-Rad), the membrane was incubated in EZ-ECL (Biological Industries) solution for 5 min.
For far western blotting, the experiment was performed similar to the western blot experiment, but CEACAM1-Ig (2 µg/ml) (Gur et al., 2019b (link)) was used instead of the first antibody and the HRP-conjugated α - human IgG (Jackson ImmunoResearch Laboratories, diluted 1:5,000) was used as a secondary antibody.

Samples were prepared as described above. The supernatant was separated by SDS gel electrophoresis in TGX Stain-Free FastCast 12% Acrylamide Gels (Biorad). After separation, the Stain-Free visualization was enabled by activation of the gels for 45 seconds with UV-light before the proteins were transferred by Trans-Blot Turbo Transfer (Biorad) onto a nitrocellulose membrane (Trans-Blot Turbo RTA, Biorad). A mouse anti-His antibody (Biorad) was used as a primary antibody in a 1:500 dilution. A goat anti-mouse IgG StarBright Blue 700 fluorescent antibody (Biorad) was used as a secondary antibody in a 1:2500 dilution. Fluorescence signals and Stain-Free Imaging were detected in a ChemiDoc MP Imaging System (Biorad). The fluorescence signals were quantified by normalization to the total protein amount detected by Stain-Free visualization, after determining the linear range using Image Lab (Biorad).