agalactiae PG2T cell lysates. The best results were obtained by means of Triton X-114 fractionation. Figure 1A illustrates the hydrosoluble and liposoluble
fractions obtained from M. agalactiae PG2T, flanked by the total protein Salubrinal cost pattern for comparison. The efficiency of the procedure in separating liposoluble proteins was evaluated by Western immunoblotting using a rabbit Selleckchem Veliparib hyperimmune serum raised against M. agalactiae P48, a previously characterized surface lipoprotein [12, 19]. As expected, presence of P48 was observed only in the total extract and in the Triton X-114 phase (Figure 1B), confirming that the fractionation method enabled separation and enrichment of hydrophobic proteins. Figure 1 Total protein patterns and Western immunoblotting reactivity of M. agalactiae PG2 T proteins. Panel A. Coomassie blue staining. Panel B: Immunoblotting reactivity obtained with antibodies against the P48 lipoprotein. From left to right: M: molecular weight standards in kDa; T: total protein
pattern; H: hydrosoluble protein fraction; L: liposoluble protein fraction obtained after Triton X-114 fractionation 2-D PAGE/MS of M. agalactiae PG2T liposoluble selleck screening library proteins Total proteins and the Triton X-114 soluble fraction of M. agalactiae PG2T were subjected to 2-D PAGE separation in order to evaluate the extent of enrichment in basic and liposoluble proteins. As illustrated in Figure 2, left panel, a very high number of spots were present in the total protein map of M. agalactiae
PG2T but, as expected, basic proteins were poorly represented. Upon comparison, the 2-D PAGE map generated with the Triton X-114 soluble fraction showed a significant enrichment in basic proteins, with an excellent resolution also in high-abundance spots (Figure 2, right panel). Figure 2 2-D PAGE patterns of M. agalactiae PG2 T protein extracts. Left: 2-D PAGE of a M. agalactiae PG2T total protein extract. Right: 2-D PAGE of M. agalactiae PG2T liposoluble proteins obtained after Triton X-114 fractionation. Bay 11-7085 In order to attain a systematic characterization of the liposoluble proteome, the Triton X-114 phase fraction of M. agalactiae PG2T was subjected to 2-D PAGE under three different pI intervals: 3-10NL, 7-11, and 4-7 (Additional files 1, 2, and 3). From these 2D maps, about 300 spots were excised and identified by MALDI-TOF and nanoHPLC-nanoESI-Q-TOF MS. This approach led to the successful identification of 40 unique proteins, corresponding to 5.4% of all M. agalactiae PG2T genes. Figure 3 reports a representative liposoluble protein map summarizing the main protein identifications accomplished on 2-D spots. A detailed description of all protein identifications is given in Additional file 4.