Monoclonal anti his tag antibody

After expression, E. coli cells were harvested by centrifugation. Lysis buffer (50 nM Tris–HCl, 0.05 mg/mL lysozyme, 1 mM PMSF, pH 7.4) was added to resuspend the cell pellet, yielding a 10% (w/v) suspension. After overnight incubation at −20 °C, sonication was applied, and the supernatant was collected. Based on the confirmation of successful expression of the IA-2ic-3LysM fusion protein by 10% SDS-PAGE and Western blot using the monoclonal anti His-tag antibody (Sangon Biotech, Shanghai, China) as the primary antibody (1:2000 dilution), purification of the IA-2ic-3LysM fusion protein from the harvested sample was performed using a Ni-NTA column (Thermo Fisher, Waltham, MA) following the manufacturer's instructions. Eluted IA-2ic-3LysM fusion protein was pooled and dialyzed against phosphate buffer saline (PBS, pH 7.0), and then concentrated using a 30-kDa cutoff filter (Millipore, Billerica, MA). Protein concentration was analyzed via Bradford Protein Assay (Bio-Rad, Hercules, CA).

0.3 volume PBS was used to wash the harvested stationary-phase L. lactis MG1363 cells. After washing two times, cell pellets were boiled for 30 min in 0.2 volume 10% trichloroacetic acid (TCA). After vigorous washing, the obtained MG1363 BLPs were resuspended in PBS (2.5 × 10¹⁰ BLPs/mL). Different amounts of purified IA-2ic-3LysM fusion protein (from 60 to 150 μg at 10 μg intervals) were added to 2.5 × 10⁹ BLPs in 1 mL PBS, and samples were then incubated for 60 min on an end-over-end rotator at room temperature. After binding, MG1363 BLPs-IA-2ic were harvested by centrifugation and the supernatant was collected and saved for Western blot to analyze whether there was some IA-2ic-3LysM residue in the supernatant after binding. The harvested pellets were washed twice with PBS and finally resuspended in 100 μL PBS. Using the monoclonal anti His-tag antibody and FITC-conjugated IgG secondary antibody (Sangon Biotech, Shanghai, China), immunofluorescence microscopy was applied as previously described (Mao et al., 2017 (link)) to confirm the successful binding of IA-2ic-3LysM on the surface of BLPs.