Zheng et al. BMC Genomics 2011, 12:40 http://www.biomedcentral.com/1471-2164/12/40 extracted membrane fractions (see Methods for details) were treated with 2% (w/v) Triton X-100 to a final con- centration of 0.2% and 0.1%, respectively, while another two samples were resolved with DDM to a final concen- tration of 2% and 1% (w/v), respectively. The migration of each sample treated with different detergents and concentrations was compared and the best result was obtained with 0.2% (w/v) of Triton X-100 (Figure 1). This concentration appeared to represent a good com- promise, avoiding both the partial disintegration of native complexes caused by excess detergent, and the appearance of gel band smearing due to insufficient concentrations of detergent. Separation by BN PAGE and SDS PAGE In the present study, biological membranes were solubi- lized with 0.2% (w/v) Triton X-100 and separation by BN PAGE. According to their size, proteins migrated more slowly with increased running distance and decreased pore size of the gradient gel and stopped almost entirely when they approached their size- dependent specific pore-size limit [16]. Figure 2 shows a representative BN PAGE map of membrane fractions. Figure 1 BN PAGE of membrane fractions from M. bovis BCG. Membrane fractions were incubated with detergents for at least one hour at 4°C. Lane 1 was High Molecular Weight (HMW) calibration and lane 2, 3 were treated with Triton X-100 (0.2% and 0.1% (w/v), respectively). Lane 4, 5 were solubilized with DDM (2% and 1% (w/v), respectively). Gel gradient was 4-16%. The gel was run according to the protocol given in Methods. Page 3 of 11 Figure 2 BN PAGE separation of M. bovis BCG membrane fractions treated with 0.2% Triton X-100. Gel gradient was 4-16% and the gel was stained with CBB G-250. Left lane was High Molecular Weight (HMW) calibration, and right was migration of BCG membrane fractions. To determine the protein composition in BN gel bands, nine bands with high staining intensity were excised and the peptide mixtures derived from them were individu- ally subjected to LC-MS/MS system as described in the experimental procedures. Finally, proteins were identi- fied by database search and replicate measurements have confirmed the identity of these protein hits. The result showed that each BN band represented a pro- tein complex. Totally, nine potential multiprotein com- plexes were obtained. The blue band I and band VII contained some subunits of lipid metabolism complex, whereas the sharp band II was identified as ATP synthase. In band III and IV, two members of phenolpthiocerol synthase and three of Emb protein complex were found, respectively. In band V two polyketide synthases were recognized, whereas band VI contained four members of heat shock protein family. In addition, several members of polyribosomal proteins were found in band VIII. In the last band IX, two subunits of the succinate dehydrogenase (SDH) enzyme complex were identified. In 2008, Målen et al. obtained a comprehensive pic- ture of the M.bovis BCG membrane protein repertoire [12]. Of these proteins identified in their study, four