Bugbuster protein extraction buffer

Overnight cultures were used to inoculate each strain into the appropriate prewarmed medium to an initial OD₆₀₀ of 0.05. The strains were grown with increasing concentrations of Aurodox to an OD₆₀₀ of 0.7 to 0.9 before being centrifuged at 3,800 rpm for 10 min to separate the cell mass. Cell lysates were prepared using BugBuster protein extraction buffer (Merck, NJ). The supernatant was removed, and proteins were precipitated by the addition of trichloroacetic acid to a concentration of 10% and incubated overnight at 4°C. The suspension was centrifuged at 3,800 × g for 1 h to form a protein pellet which was suspended in 100 µl of Tris-HCl (pH 8.0). The samples were then mixed 1:1 with loading dye (Novex, Waltham, MA) and run on a 4 to 12% SDS-PAGE gel at 120 V for 1 h. Gels were subsequently stained with Coomassie brilliant blue stain (Novex) and destained in water overnight. When required, bands were excised for subsequent in-gel digestion and analysis. Proteins analyzed by tandem mass spectrometry were given a MASCOT score to indicate the probability of the identification being correct.

SDS-PAGE analysis of secreted proteins was carried out as described previously [51 (link)]. Strains of interest were cultured in 50 ml MEM-HEPES as above to an OD⁶⁰⁰ of ~0.8 and supernatants were obtained by centrifugation at 4000 rpm for 20 minutes. Whole cell pellets were lysed using BugBuster Protein Extraction buffer (Merck, New Jersey, USA). For secreted proteins, cell culture supernatants were syringe filtered (0.45 μm) and precipitated with 10% v/v TCA (Sigma) overnight at 4°C. Secreted proteins were harvested by centrifugation at 4000 rpm (4°C) for 1 hour. Protein pellets were resuspended in Tris-HCl (pH 8.0) and analyzed by SDS-PAGE using the Novex system (Invitrogen, Carlsbad, CA, USA). Primary antibodies used for immunoblotting were EspD (1/6000), Tir (1/2000) and GroEL (1/20000). Antibodies were made up in PBST containing 1% skim milk powder. Comparison of protein levels from SDS PAGE and immunoblot experiments was carried out by densitometry using ImageJ. Experiments were performed on multiple occasions to confirm the results.