Anti groel polyclonal antibodies

Protein lysates were prepared in SDS–PAGE sample buffer. Proteins were separated by SDS–PAGE in 12% polyacrylamide gels and electrophoretically transferred to nitrocellulose filters for Western blot analysis using anti-Flag M2 monoclonal antibodies (1:5000; Sigma) or anti-GroEL polyclonal antibodies (1:30,000; Sigma). Goat anti-mouse IgG IRDye 800CW and goat anti-rabbit IgG IRDye 680RD (LICOR) were used as secondary antibodies. Bands were detected using and Odyssey Fc infrared imaging system (LICOR).

P. aeruginosa cultures grown in LB supplemented with 20 mM Mg₂Cl + 5 mM EGTA, up to an OD₆₀₀ 1.5, were used to detect ExoS and ExoU. Proteins from supernatants were precipitated with trichloroacetic acid and separated by 15% SDS-PAGE electrophoresis, transferred onto a nitrocellulose membrane, and probed by Western blot assays using anti-ExoS or anti-ExoU polyclonal antibodies, as reported previously [51 (link)]. Anti-GroEL polyclonal antibodies (Sigma) were used to detect the GroEL protein as a load control. Blots from membranes were developed with the HRP luminol Super Signal chemiluminescent substrate (Thermo Scientific).