Sucrose density gradient centrifugation and dot blot

The supernatant of 3TC-treated HepAD38 cells was mixed with PEG-8000 powder (final concentration (wt/vol) of 10%) and gently rotated at 4°C overnight, HBV particles were then precipitated by centrifugation at 1,000×g for 30 min at 4°C and re-dissolved in 1×TNE buffer. Gradients were formed by overlaying 3 ml of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, and 55% (wt/vol) sucrose solutions in TNE buffer and incubated at 4°C for 10 h to allow gradient to become continuous. Then, five milliliters of the prepared HBV particle solution were overlaid on the gradients. Samples were centrifuged in Beckman SW28 rotor at 26,000 rpm for 16 h at 4°C. Twenty-two fractions of approximately 1.7 ml were collected from the bottom of the tube.

To detect HBcAg and HBsAg, 100 μl of each fraction was dot-blotted on nitrocellulose membrane and probed with the corresponding antibody (Dako), the bound antibody was detected with IRDye secondary antibodies by using Li-COR Odyssey Fc System. For detection of particle-associated HBV DNA, the dot blot membrane was soaked in denaturing solution containing 0.5 N NaOH and 1.5M NaCl for 15min, followed by neutralization with a solution containing 1 M Tris-HCl (pH7.6) and 1.5 M NaCl for 5 min. HBV DNA was detected by hybridization with an [α-³²P]-UTP-labeled HBV minus-strand-specific riboprobe. For detection of particle-associated HBV RNA, the dot blot membrane was transiently treated by the above-mentioned denaturing buffer for 20 s, followed by neutralization for 5 min. HBV RNA was detected by hybridization with an [α-³²P]-UTP-labeled HBV plus-strand-specific riboprobe.