Zheng et al. BMC Genomics 2011, 12:40 http://www.biomedcentral.com/1471-2164/12/40 This observation may suggest that some pigment mole- cules can bind to gel without destaining completely. Conclusions In the present study, by coupling LC-MS/MS to BN PAGE we were able to unambiguously identify potential membrane/membrane-associated protein complexes in BCG membrane fractions. The proteins identified in complexes were confirmed using 2-D gel. For example, eight subunits of ATP synthase complex which were obtained in BN gel band were all detected in 2-D gel. Furthermore, we were able to identify large, extremely hydrophobic proteins in BN gel by in-gel digestion and MS/MS analysis. In summary, this study is the first to apply the BN PAGE technique to the separation of potential BCG membrane protein complexes. Our results may provide some clues for BCG protein interac- tion studies and designing strategies against bacterial infection. Methods Bacterial cell cultivation M. bovis var BCG NCTC 5692 from Beijing Tuberculo- sis and Thoracic Tumor Research Institute (Beijing, P.R. China) was cultivated in 5 liters of Sauton’s liquid med- ium for 3 weeks at 37°C without shaking. Cells were harvested by centrifugation at 12,000 × g for 10 min at 4°C, and washed three times with ice-cold phosphate buffered saline (pH 7.4). Protease inhibitor cocktail (Roche, Germany) was added to all buffer solutions before use. The pelleted cells were frozen at -80°C prior to use. Sample preparation All bacteria and sample manipulations were performed at 4°C. Membrane samples were prepared according to the method described by Sinha et al. with some modifi- cations [7]. In brief, the cell pellet was probe-sonicated in ice-cold sonication buffer (50 mM Tris-HCl, l0 mM MgCl2, 0.02% sodium azide, pH7.4) containing protease inhibitor cocktail. The sonicates were centrifuged initi- ally at 20,000 × g for 20 min to remove any unbroken cells and debris, and later at 150,000 × g for 90 min to obtain cell membrane sediment. The sediment was washed three times in sonication buffer containing pro- tease inhibitor to remove cytosolic contaminants. The washed sediment was divided into four aliquots. The first two aliquots were suspended in sample buffer (750 mM ε-aminocaproic acid, 50 mM Bis-Tris, 0.5 mM EDTA-Na2, pH 7.0) and 10% DDM was added to a final concentrations of 2% and 1%, respectively. The samples were stirred slowly on ice for at least 1 h to solubilize the membrane fractions. The soluble proteins were col- lected by centrifugation at 12, 000 × g for 60 min at 4°C Page 8 of 11 prior to electrophoresis. The other two aliquots were solubilized with 2% Triton X-100 to final concentrations of 0.2% and 0.1%, respectively. The subsequent samples were treated as described above prior to electrophoresis. BN PAGE and extraction of the peptide mixtures Membrane samples were analyzed by BN PAGE accord- ing to the method of Schägger and von Jagow with minor modifications [17]. Briefly, a stacking gel of 3.5% and a separating gradient gel of 4-16% were used. Anode and cathode electrophoresis buffers were differ- ent as described previously [44]. About 80 μg protein from each aliquot was loaded to each lane and electro- phoresis was conducted at 120 V for 2 h, and then adjusted to 200 V. After the blue running front has moved about half the desired total running distance, the blue cathode buffer was replaced with colorless BN cathode buffer for better detection of faint protein bands and the run was then continued until the front had reached the bottom of the separating gel. A high molecular weight calibration kit (GE Healthcare, UK) was used to indicate the protein size. Thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa) and bovine serum albumin (66 kDa) were used as markers. After electrophoresis, gels were stained with colloidal Coomassie stain as described previously [45]. From the stained gels, nine bands with high staining intensity were chosen for excision and destaining. Proteins in each band were reduced with 10 mM dithiothreitol and alkylated with 55 mM iodoacetamide. In-gel trypsin digestion was then performed as described previously [46]. Tryptic peptides were then extracted by incubating each band with extraction solution (50 μl 1% TFA in 60% ACN) for 20 min at RT. The supernatant collected by centrifugation were concentrated and desalted with ZipTip C18 (Millipore, USA). The tryptic peptides were dried using a Speed-vac centrifuge (Eppendorf, Germany) and resolubilized in 0.1% TFA for subsequent 2-D LC-MS/MS analysis. LC separation and MS identification Each distinct sample of 8 μl peptide mixture was loaded by the autosampler onto a reverse phase C18 PepMap 100, 3 μm, 100Å nano-column (75 μm i.d. × 15 cm) of Ultimate 3000 Nano and cap system (Dionex, USA). The flow rate was 2.0 μl/min for the nano-column, and the solvent gradient was 4% B to 60% B in 30 minutes, then 60-100% B in 5 minutes. Solvent A was aqueous 0.05% TFA and solvent B was aqueous 80% ACN in 0.04% TFA. Thirty seconds fractions of the LC flow were automatically spotted onto a Prespotted AnchorChip disposable targets using PROTEINEER fc (Bruker, Germany). Dried samples were washed with