Secondary anti mouse igg

HSC-2, SAS, and Ca9-22 cells (2 × 10⁵) were resuspended in 100 μL of serially diluted PcMab-47, 47-mG_2a, 47-mG_2a-f, or chPcMab-47 (6 ng/mL to 100 μg/mL), followed by the addition of secondary anti-mouse IgG (Cell Signaling Technology) or anti-human IgG (Thermo Fisher Scientific). Fluorescence data were collected using a cell analyzer (EC800). K_D was obtained by fitting the binding isotherms using the built-in one-site binding models in GraphPad PRISM 6 (GraphPad Software, La Jolla, CA, USA).

The expression of surface streptavidin was first detected by western blotting. Secreted streptavidin proteins in the Lp culture were precipitated with 15% trichloroacetic acid (TCA), separated via SDS-PAGE, and transferred onto a 0.22μm nitrocellulose membrane (Bio-Rad). The transferred proteins were visualized on the western blot through detection by a primary c-Myc antibody at a 1:1000 dilution in 3% BSA and a secondary anti-mouse IgG (Cell Signaling #7076) at a 1:1000 dilution in 3% BSA. The membrane-bound streptavidin proteins were dissolved in 8 M urea and visualized using an anti-HA antibody (Thermo Fisher 26183) at a 1:1000 dilution in 3% BSA and the same secondary anti-mouse IgG antibody.
After the confirmation of protein expression, engineered Lp strains were incubated with c-Myc monoclonal or HA tag antibody (1:500 dilution in 1% BSA) for one hour, followed by a one-hour incubation with the secondary anti-mouse antibody (1:500 dilution in 1% BSA) at 25 °C. The antibody-treated cells were then visualized by a Leica DM4000 B fluorescence microscope or analyzed in the CytoFLEX analyzer.