In brief, 3-4 week old bacilli were lysed by bead beating and centrifuged, initially at 2300 g to remove unbroken cells and cell-wall debris. Triton X-114 was added to the supernatant (final detergent concentration 2%, v/v) and the suspension was stirred at 4°C for 20 minutes to obtain the protein extract in a single phase. Residual insoluble matter was removed by centrifugation at 15700 g for 10 min, and the solution separated into two phases, an upper (aqueous) and lower (detergent) phase after 10 minutes incubation at 37°C. The detergent phase was collected and proteins were precipitated by acetone. Gel electrophoresis and in-gel digestion of proteins Extracted

proteins (50 μg) were mixed with 25 μl SDS loading buffer and boiled for 5 minutes before separation on a 10 cm long 1 mm thick 12% SDS polyacrylamide gel (Invitrogen, Carlsbad, CA, U.S.A.). The protein migration was allowed to proceed until the bromophenol dye had https://www.selleckchem.com/products/tideglusib.html this website migrated to the bottom of the gel. The protein bands were visualized with Coomassie Brilliant Blue R-250 staining (Invitrogen).

Protein lanes were excised and divided in fractions according to the bands of the protein standard, ranging from ~3 kDa to ~188 kDa. The gel pieces were washed twice with 50% acetonitrile (ACN) in 25 mM ammonium bicarbonate (NH4HCO3) for 15 minutes at room temperature (RT), and subsequently dehydrated by incubating them with 50 μl 100% ACN for 20 minutes at of RT. The proteins were reduced using 10 mM dithiotreitol and alkylated with 55 mM iodoacetamide; both in 100 mM NH4HCO3. The gel pieces were dehydrated by 100% ACN as described above, and rehydrated in 25 mmol/l NH4HCO3 followed by in-gel protein digestion with trypsin (Promega, Madison, U.S.A.) for 16-20 h at 37°C. The digested peptides were eluted by incubating the gel pieces with 50 μl 1% formic acid (FA) for 20 minutes at RT. The supernatant containing the peptides were collected after centrifugation at 15700 g for 10 minutes. Then, the gel pieces were incubated with 50 μl 0.1% FA in 50% ACN for 20 minutes at RT, followed by centrifugation

at 15700 g. The supernatant was collected and combined with the previous one. Finally, the gel pieces were dehydrated with 50 μl 100% ACN for 20 minutes at RT, and the supernatant was collected after centrifugation as described above and added to the pool. Mass spectrometry Experiments were performed on a Dionex Ultimate 3000 nano-LC system (Sunnyvale CA, USA) connected to a linear quadrupole ion trap-Orbitrap (LTQ-Orbitrap) mass spectrometer (Thermo Electron, Bremen, Germany) equipped with a nanoelectrospray ion source. The mass spectrometer was operated in the data-dependent mode to automatically switch between Orbitrap-MS and LTQ-MS/MS acquisition. Survey full scan MS spectra (from m/z 400 to 2,000) were RAD001 cost acquired in the Orbitrap with resolution R = 60,000 at m/z 400 (after accumulation to a target of 1,000,000 charges in the LTQ).