Bacterial Protein Extraction and Purification

Cell lysis, protein extraction, and purification were performed essentially as described previously [20 (link), 21 (link)]. Briefly, bacterial cells were lysed with B-Per extraction reagent (Thermo Scientific, #78248) supplemented with DNAse I (Thermo Scientific, #90083) and protease inhibitor cocktail (Thermo Scientific, #87785) following manufacturer’s instructions. Clear supernatant containing soluble proteins was passed through 0.45 μm membrane (Millipore, #HPWP04700) and purified using HisPur Cobalt resin (Thermo Scientific, #89965) following supplier’s protocols. Purified proteins were dialyzed against PBS using Slide-A-Lyzer Dialysis Cassettes, 10K MWCO (Thermo Scientific, #66710) and stored at -80°C in small aliquots. SDS-PAGE and western blot analyses were performed following standard protocols and as described previously [20 (link)]. Intensities of immunoreactive bands on western blots were quantified by densitometric analysis performed with ImageJ software [22 (link)].