Figure 3 Identification of putative mucin binding Wnt inhibitor proteins Repotrectinib concentration by blot overlay

assay. SDS-extracted putative surface proteins were separated by a 7% SDS-PAGE and Western blotted onto nitrocellulose and incubated with purified MUC7 preparation (50 μg/ml). Binding of MUC7 to putative surface proteins determined by immunological procedures probing the membrane with AM-3 antibody and ECL detection (B). Molecular masses of the MUC7-binding proteins were calculated in Bio-rad model GS-700 imaging densitometer and it’s PC compatible software. A control Western blot, which had been incubated with PBS instead of MUC7 preparation was probed with AM-3 antibody and subjected to ECL detection (C). The efficiency of the Western transfer of the separated SDS-extracted proteins was assessed by amido black staining of the membranes (A). Positions of the molecular weight markers are indicated (kDa). Results are shown as one representative experiment of multiple independent preparations. Further characterization of the MUC7-binding proteins required their preparative separation and purification; hence, the SDS-extracted proteins from intact S. gordonii

were fractionated by preparative SDS-PAGE and the resulting fractions were analyzed by analytical SDS-PAGE (Figure 4). The electrophoretic analysis of the selected fractions indicated that putative MUC7-binding bands could CBL0137 datasheet be separated from other streptococcal proteins (Figure

4A). This separation of the adhesin bands from the nearest contaminant allowed a cleaner sample for in-gel digestion and subsequent protein identification. In order to determine the fractions that contained MUC7 binding proteins, aliquots of the fractions from the preparative Carnitine dehydrogenase electrophoresis were transferred to the nitrocellulose membranes by slot blotting and probed with 50 μg/ml MUC7 in PBS (Figure 4B). Antibody reactivity was detected around the fractions 12–13 (62 kDa), 20–21 (74 kDa), 24–25 (84 kDa) and 44–45 (133 kDa), confirming the result obtained from Western transfer and following overlay assay as described above. Figure 4 Preparative SDS-PAGE of SDS-extract from Streptococcus gordonii PK488 and identification of MUC7 binding proteins. Twenty milligrams of the surface extract from S. gordonii was electrophoresed on a 7.5% preparative electrophoresis in a Bio-Rad mini-prep cell and (A) selected fractions were electrophoresed on 7.5% SDS-PAGE gels, proteins visualized with silver stain. (B) Selected fractions were transferred onto nitrocellulose membranes by slot blotting and probed with MUC7 preparation. MUC7 binding was determined by immunoblotting as described in Material and Methods. Positions of molecular weight markers are indicated (kDa). Putative adhesin bands were subjected to in-gel digestion and the resultant peptides were analyzed by LC-MS/MS.