Fig. 1. Characterization of the I/F8 antigen. (A) Immunoprecipitation from Sulfo-NHS-LC-biotin surface-labeled IGROV-I tumor cells using the I/F8 scFv coupled to CNBr-activated Sepharose beads. Immunoprecipitates were resolved by SDS-PAGE on a 10% polyacrylamide gel, and revealed by western blot analysis using HRP-conjugated Streptavidin and chemiluminescence. The I/F8 antigen is a 116 kDa monomer under both reducing (ME+) and non-reducing (ME–) conditions. Immunoprecipitates using an irrelevant scFv coupled to Sepharose beads were run as negative controls (ctr). (B) Silver-stained preparative immunoprecipitation and SDS-PAGE analysis of the I/F8 antigen. I/F8 immunoprecipitates from several tumor cell lines were resolved by SDS-PAGE on a 10% polyacrylamide gel under reducing conditions, and revealed by staining with silver nitrate. Bands were then excised from the gel and processed for MALDI-TOF analysis. (C) Immunofluorescence analysis of I/F8 (black line) and J4-81 (gray line) reactivity on various human and murine tumor cell lines. Broken line represents negative control with an irrelevant scFv. (D) Silver staining of I/F8 immunoprecipitated molecules from three murine tumor cell lines, resolved by SDS-PAGE on a 10% polyacrylamide gel. Numbers on left-hand side of blots indicate molecular weight markers in kDa.