3 M NaCl before being resuspended in 0.2 ml of 0.03 M Tris-HCl (pH 8.0), 20% (wt/vol) sucrose, and 0.1 mM EDTA at 25°C. After Temsirolimus supplier 10 min the cells were pelleted and rapidly suspended in 0.3 ml of ice-cold 0.5 mM MgCl2. After incubation on ice for 10 min, the cells were removed by centrifugation at 12,000 × g. The supernatant was used

as the periplasmic protein extract. The cell pellet was then disrupted by sonication in 0.5 ml ice-cold water. The cell debris and unbroken cells were removed by centrifugation at 5,000 × g for 10 min at 4°C, and the next supernatant was fractionated into the membrane and cytoplasmic fractions by centrifugation at 10,000 × g for 30 min at 4°C. The resulting supernatant was used as a cytoplasmic fraction. The sediment was resuspended in sterile distilled water and used as the membrane fraction. In order to separate the inner and outer membranes, the membrane fraction was further treated with N-lauryl sarcosyl at a final concentration of 2% at room temperature and then centrifuged at 15,000 × g for 30 min. The resulting sediment was used as the outer membrane fraction, and the supernatant was used as the inner membrane fraction after dialysis and precipitation. Extracellular, periplasmic, cytoplasmic, and membrane-bound proteins were concentrated by precipitation with ice-cold trichloroacetic

acid (final concentration, 10%). The precipitated proteins were collected by centrifugation at 12,000 × g, washed with acetone, dried under vacuum, and dissolved in sample buffer (50 mM Tris-HCl [pH 6.8], 10% glycerol, 5% β-mercaptoethanol, 2% sodium dodecyl sulfate [SDS], 0.05% bromophenol blue). Samples were neutralized by learn more addition of a saturated Tris solution and boiled for 5 min at 100°C before SDS-PAGE analysis. The amount of sample from each

extract used for the SDS-PAGE was as follows: 2.5 μl of the 150 μl STK38 cytoplasmic (C) extract; 2.5 μl of the 40 μl inner membrane (IM) extract; 5 μl of the 100 μl periplasmic (P) extract; 2.5 μl of the 40 μl outer membrane (OM) extract and 2.5 μl of the 300 μl whole cell (WC) extract. In all cases the extracts were derived from 1 ml culture samples and the relative amount of the extracts used for SDS-PAGE in comparison with the amount of WC extract used (arbitrarily set to 1.0) were 2 × for C; 8 × for IM; 6 × for P, and 8 × for OM. SDS-PAGE and N-terminal sequencing SDS-PAGE was performed using the method described by Laemmli [36]. Proteins were blotted onto PVDF membrane and stained with Coomassie brilliant blue. 50% methanol was used for de-staining the membrane to visualize the protein bands. Proteins present in visible bands were excised from the membrane for N-terminal sequencing. Determination of the N-terminal amino acid sequence of proteins was achieved by automated Edman degradations of samples blotted onto PVDF membranes. The sequencing was performed on a Procise 494 Sequencer (Applied Biosystems) with an on-line PTH-analyzer.