Immobilon western chemiluminescent hrp substrate ecl working solution

Proteins extracted from cells or concentrated from elutriants were analyzed with SDS-PAGE, the proteins on the membrane were probed with anti-HCV Core (C7-50, Abcam Ltd.), anti-NS3 (H23, Abcam Ltd.), anti-hA3G (ab75560, Abcam Ltd.), anti-HA (6E2, Cell Signaling Biotechnology Inc.), or anti-HA-tag [HRP] (A00169, GenScript.) antibody, respectively, with anti-Actin antibody (TA-09, ZSGB-BIO, China) served as the control. After washing with TBST, the membrane was incubated with goat anti-mouse (sc-2005, Santa Cruz Biotechnology Inc.) or goat anti-rabbit (sc-2004, Santa Cruz Biotechnology Inc.) secondary antibody, respectively. Protein signals were visualized and captured using Immobilon Western Chemiluminescent HRP Substrate ECL working solution (Millipore Inc.) with ChemiDo XRS gel imager system (Bio-Rad, CA).

The extracted total protein or viral lysates were denatured by adding loading buffer (5 × 250 mM tris-HCl, pH 6.8, 5% dithiothreitol, 10% SDS, 0.5% bromophenol blue, 50% glycerol), followed by boiling the lysates for 5 minutes at 100 °C. Proteins were analysed by SDS-PAGE and then transferred to nitrocellulose membranes using an electroblotter (Bio-Rad Laboratories). The membranes were blocked by 5% non-fat dry milk in TBS-T solution (20 mM Tris [pH 7.4], 150 mM NaCl, 0.1% Tween-20) for 1 hour and then washed three times for 10 minutes each in the TBS-T solution. Membrane samples were probed with a monoclonal antibody specific to the HCV core protein (diluted to 1 μg/mL; Abcam, Ltd.) or Hsc70 (diluted to 0.2 μg/mL; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). As a control, probing experiments were performed using a polyclonal antibody against actin (diluted to 0.2 μg/mL; Santa Cruz Biotechnology, Inc.). After being washed with TBS-T, the membranes were incubated for one hour at room temperature with the appropriate secondary antibody: goat anti-mouse for the HCV core protein, goat anti-rat for Hsc70, and goat anti-rabbit for actin (ZSGB-BIO, China). The protein signal was visualized and captured using Immobilon Western Chemiluminescent HRP Substrate ECL working solution (Millipore Inc.) and a ChemiDoc™ XRS gel imager system (Bio-Rad, CA).