Hrp conjugated donkey anti rabbit igg

Protein samples were prepared from equal amounts of bacteria cells after overnight growth at 30°C. Bacteria were harvested by centrifugation at 10,000×g for 10 min at 4°C. The culture supernatant fluid was precipitated with 10% trichloroacetic acid (TCA). Briefly, 1 volume (250 µl) of 50% TCA stock was added to 4 volumes (1 ml) of protein sample. The protein-TCA mixture was kept on ice for 15 min, and subsequently the tube was centrifuged at 15,000×g for 5 min. The supernatant was removed and the protein pellet was washed with 200 µl of cold acetone. Finally, the tube was centrifuged at 15,000×g for 5 min and the resulting pellet was dissolved in sample buffer containing 10% glycerol, 0.05% bromophenol blue, 2% SDS, 5% 2-mercaptoethanol, and 10 mM Tris-HCl, pH 6.8. Proteins with known molecular masses (Fermentas) were used as molecular mass markers. SDS-PAGE and Western blotting were carried out according to the methods of Laemmli [56] (link) and Towbin et al. [57] (link). HRP-conjugated donkey anti-rabbit IgG (Promega, USA) was used as secondary antibody. Detection was performed using ECL Prime Western Blotting Detection Reagent (Amersham or GE Life Sciences, USA). Pre-stained Protein Ladder (SM0679, Fermentas) was used as size standards. Gels were stained with Coomassie brilliant blue.