This protein band was subjected to in-gel digestion and the resultant peptides were analysed by LC-MS/MS. Three peptides (SHFELPHYPGLLAHQKPFIR, LPPSPNNPPK, and

FLLYMK) from the MUC7 core protein were clearly identified by mass spectrometry. The gel was also transferred check details to nitrocellulose membranes and probed with the AM-3 monoclonal antibody. AM-3 reactivity showed one distinct band at the same region with Coomassie blue stained protein which was later identified as MUC7 (Figure 1B). Figure 1 SDS-PAGE and Western blot analysis of purified MUC7 preparation. MUC7 purified by employing a two-step chromatographic protocol as described in Methods. (A) Final purified MUC7 pool from Mono Q HR 10/10 ion exchange column was electrophoresed

in a Midget 7.5% SDS-PAGE gel under reducing conditions and visualized by Coomassie blue staining and Western transferred Afatinib to nitrocellulose membranes and probed with AM-3 monoclonal antibody (B). Positions of the molecular weight LY2606368 manufacturer markers are indicated (kDa). Extraction and separation of SDS-extracted Streptococcal surface proteins SDS-extracted proteins from intact S. gordonii were separated by SDS-PAGE under non-reducing conditions (Figure 2). The extract yielded a large number of bands; at least 30 bands were observed on the gel. In order to check for possible cell lysis and hence contamination by intracellular proteins, the extract was examined for presence of DNA by UV spectrophotometry but none was detected (260/280 ratio was smaller than 0.6, data not shown). Figure 2 Protein profile of SDS-extracted surface proteins from S. gordonii: 10 μg of the SDS-extract supernatant from S. gordonii was electrophoresed on a 10% SDS-PAGE gel under non-reducing condition. L-gulonolactone oxidase Separated proteins

were stained by Coomassie blue. Positions of the molecular weight markers are indicated (kDa). Results are shown as one representative experiment of three different S. gordonii preparations. Identification of Putative MUC7 binding proteins by blot overlay assay In order to identify streptococcal proteins that bind MUC7, the SDS-extracted proteins were Western blotted onto nitrocellulose membranes and incubated with the MUC7 preparation. Mucin binding was quantified by immunoblotting with an antibody against a glycan on MUC7. The transfer of the separated proteins to nitrocellulose membranes was assessed by a visual comparison of blots stained with amido black compared to replica SDS-PAGE gels stained with Coomassie blue (Figure 3A). The comparison shows that all bands seen in the SDS-PAGE gel (Figure 2) were represented on the membrane. The extracted and separated proteins were blotted onto nitrocellulose and subsequently incubated with purified MUC7 (50 μg/ml) preparation. Detection of bound MUC7 with monoclonal antibody AM-3 identified several putative adhesin bands with apparent molecular mass 62, 78, 84, 133 kDa (Figure 3B).