Protein extracts were obtained from 140 mL of M. tuberculosis mid-log cultures grown in 7H9–0.05% Tween 80 supplemented with 10% ADC. Cells were pelleted by centrifugation and washed twice with sterile PBS 1X. Pellets were resuspended in lysis solution (7M Urea, 2 M Thiourea, 4% CHAPS, 4% ASB-14) and then sonicated (20 cycles: 30 sec ON/1 min OFF) at 4 °C in ice water, and finally centrifuged to remove cells debris. Samples were then quantified and purified using 2-D Quant Kit and 2-D Clean-Up Kit (GE Healthcare) respectively. For two-dimensional gel electrophoresis (2DE) IPGphor II and EttanSix (GE Healthcare) were used for the first and second dimension respectively. 100 μg of each sample was separated by 2DE in 24 cm length, 6–9 pH strips in the first dimension and in a 12% SDS-PAGE for the second dimension according to manufacturer instructions. 2DE gels were silver stained as described elsewhere31 (link). Image analysis was performed using Progenesis SameSpot software (Nonlinear Dynamics). After image analysis, differential spot was excised and identified as previously described by mass spectrometry32 (link).
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