Figure 1

Characterization of scFv N14 antibody. (A) Purification of recombinant scFv N14 antibody. 1: supernatant of cell lysates of E.coli BL21 (DE3); 2: recombinant scFv N14 antibody expressed as inclusion body; 3 and 4, purified refolding scFv N14 antibody. (B) ELISA analysis of the binding affinity of scFv N14 antibody to LO2 and HepG2 cells. scFv N14 antibody (black bar) was incubated with LO2 and HepG2 cells, PBS buffer (red bar) was used as the control. (C) The expression of scFv N14 antigen in HepG2 cells and LO2 cells. Equal amounts (20 μg) of whole cell extracts from HepG2 cells or LO2 cells were loaded onto the SDS-PAGE gel and normalized by comparing with ß-actin. (D) Two protein bands of 35 kDa and 37 kDa in the nuclear protein extract (lane N) of HepG2 cells are recognized by scFv N14 antibody. These bands are not seen in the cytoplasmic protein extract (lane C). (E) Immunofluorescence analysis of the sub-cellular localization of scFv N14 antigen within HepG2 cells. Cells were immunostained with scFv N14 antibody, mouse anti-His₆ antibody and FITC-conjugated goat anti-mouse IgG antibodies. DAPI was used to stain the nuclei. (F) The expression of scFv N14 antigen in human HCC cell lines QGY-7701, QGY-7703, SMMC-7721 and human non-cancerous liver LO2 cells. Equal amounts (15 μg) of whole cell protein extracts from various cells were loaded onto the SDS-PAGE gel and normalized by comparing with ß-actin.