Figure 3

Identification of putative mucin binding proteins 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.