Whole-cell Lysate Protein Analysis

The whole-cell lysate of L. monocytogenes F4244, P. aeruginosa PRI99, E. coli EDL933, and S. Enteritidis PT21 overnight cultures (5 mL each) was prepared by sonication (Branson, Danbury, CT, United States). Bacterial samples were sonicated in an ice bucket (three 10 s cycles at 30-s intervals) and centrifuged for 10 min at 14,000 rpm (Eppendorf) at 4°C to separate the soluble fraction (supernatant) from the bacterial debris (pellet). The protein concentration was determined by the BCA method (Thermo Fisher Scientific). Equal amounts of proteins were separated on SDS-PAGE gel (10% polyacrylamide) and electro-transferred to polyvinylidene difluoride (PVDF) membrane (Fisher Scientific) (Singh et al., 2016 (link); Drolia et al., 2018 (link)). Primary and secondary antibodies were diluted as above. Membranes were first probed with mAb-2F11 at 4°C overnight, and then with anti-mouse HRP conjugated antibody at room temperature for 1.5 h. LumiGLO reagent (Cell-Signaling Technology) was used to visualize the bands using the Chemi-Doc XRS system (Bio-Rad).