Western blott analysis to test de novo synthesis of non structural protien (NS5A) of HCV in lysates of infected Huh7.5 cells. Huh7.5 cells were infected a pool of 20 HCV positive samples. Cells were lysed as described in Materials and Methods and the lysates were resolved on 12% SDS-PAGE, blotted onto nitrocellulose membranes and hybridized to anti-NS5A monoclonal antibody as primary antibody and the color was developed enzymatically with anti mouse IgG as secondary antibody. A distinct specific band of ~ 56 kDa corresponding to the NS5A protein appeared only in infected (lane 2) but not in the uninfected (lane 1) cells indicating de novo synthesis of viral non structural proteins following infection with the virus. Lane M represents the Protein marker (Blue Ranger Pre-stained Marker Mix; Pierce, U.S.A).