Proteases inhibitor cocktail

K. pneumoniae-43816 (ATCC) (K2 strain), K. pneumoniae-396 (K1 strain) was cultured in 30 ml tryptic soy broth (Difco) for 18 hours at 37°C and then harvested. The pellet was re-suspended in 5 ml of lysis buffer [50 mM Tris pH 8.0, 10% glycerol, 0.1% Triton X-100, 100 μg/ml lysozyme (Thermo Scientific) with 1 mM EDTA, 1 mM PMSF and proteases inhibitor cocktail (Thermo Scientific). After incubation on ice for 30 min, the bacterial lysate was sonicated for 20 s (5% Duty cycle and 50% Power output) on and off for 8–10 times until sample was no longer viscous, and finally centrifuged at 10,000 rpm for 30 min at 4°C. The protein concentration in supernatant was measured by BCA protein assay (Pierce). A similar antigen preparation was performed for the following bacteria: Acinetobacter baumannii-NR-17783 (BEI), Enterobacter cloacae-NR-50391 (BEI), Proteus mirabillis-HM-752 (BEI), Enterococcus faecalis-HM-201 (BEI), Pseudomonas aeruginosa Migula (ATCC® 29260™), Streptococcus pneumoniae (Klein) Chester (ATCC® BAA-334™), and Serratia marcescens subsp. marcescens Bizio (ATCC® 13880™).

C. rodentium was cultured in 200ml LB and incubated at 37 °C, shaken at 200rpm overnight and then harvested. The pellet was re-suspended in 5ml of lysis buffer (50mM Tris pH 8.0, 10% glycerol, 0.1% Triton X-100, 100ug/ml lysozyme (Thermo Scientific), 1mM EDTA, 1mM PMSF and proteases inhibitor cocktail (Thermo Scientific). After incubation on ice for 30min, the lysate was sonicated for 20 s (5% Duty cycle and 50% Power output) on and off for 8–10 times till sample was no longer viscous and then centrifuged at 10,000rpm for 30min at 4 °C. The supernatant protein concentration was measured by BCA protein assay (Pierce).