Figure 3
Identification of scFv N14 antigen by 2D gel/Western blot/MS analysis. (A) Nuclear protein extract of HepG2 cells separated by 2-DE, probed with scFv N14 antibody. Two protein spots reacted with scFv N14 antibody were indicated by arrows in the 2-D gel at molecular weights of 37 kDa (spot up) and 35 kDa (spot down) and pI of around 9.0. After the identification, the Western blot membrane was stripped off the scFv N14 antibody and re-probed with a polyclonal goat anti-hnRNP A2/B1 antibody to prove that scFv N14 and commercial hnRNP A2/B1 could detect the same spots. (B) Peptide sequences identified from spot down by Q-TOF analysis. (C) Western blot showing scFv N14 bound with recombinant hnRNP A2. Various amounts of hnRNP A2 (Lane 1, 0.4 μg; Lane 2, 0.7 μg; Lane 3, 1.4 μg) were mixed with 20 μg the extracts of E.coli Top10 (Invitrogen) and loaded onto the SDS-PAGE gel. ScFv N14 antibody can specifically recognize recombinant hnRNP A2 protein.