Target antigens applied to screen yeast display library. A. Schematic diagram of the primary structure of two antigen approaches selected from hGPC3 protein. The 29mer hGPC3530-558 peptide and truncated hGPC3 fused with GST are represented by gray regions. Two glycosaminoglycan binding site (Gag) and putative glycosylphosphatidyl-inositol (GPI) anchor regions within the C-terminal hydrophobic region of hGPC3 are shown. B. SDS-PAGE gel stained with Coomassie brilliant blue showing the expressed GST-fusion protein. BL21 bacteria transformed with the plasmid pGEX-4T/GPC3, encoding a GST-human/mouse GPC3 fusion protein, were induced to express the recombinant protein in presence of IPTG. Recombinant proteins were purified by glutathione agarose beads. The purified proteins (10 ul/each) were electrophoresed on a 10% SDS-PAGE gel for analysis. C. Confirmation of the purified recombinant protein by western blot. The purified recombinant protein were subjected to 10% SDS-PAGE and transferred to a nitrocellulose filter. The filter was probed with a commercial monoclonal anti-human GPC3 antibody (clone 1G12). Note the cross-reactivity of murine GPC3 with 1G12 antibody.