Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment

SV24 total cell lysate corresponding to 1.5 nag protein as measured by Bio-Pad protein assay (Bio-Rad Laboratories, Richmond, CA) was loaded into a 18-cm well and separated by electropboresis on an SDS-polyacrylamide get (10%). The proteins were then electrotransferred from the gel to a nitrocellulose sheet (Schleicber and Schuell, Dassel, Germany) using a Genie blotter apparatus (Idea Scientific Company, Corvallis, OR) cooled with ice for 1 h at 24 V constant voltage. Proteins were fixed to the nitrocellulose in 3% TCA containing 0.2% Ponceau S protein stain (Serva, Heidelberg, Germany). Strips cut from the nitrocellulose were blocked with 10% (wt/vol) fat-free instant milk powder in PBS (blotto; Johnson et al., 1984) and incubated with primary antibodies diluted in blotto. After washing with 0.2% Triton X-100 in PBS the nitrocellulose strips were incubated with alkaline phosphatase-conjugated secondary antibodies diluted in blotto. After washing with 0.2% Triton X-100 in PBS, the strips were incubated with alkaline phosphatase substrate solution (100 mM Tris-Cl, pH 9.5, I00 mM NaCI, 5 mM MgCI~, 0.33 t~g/mi nitroblue tetrazolium, 0.165 #g/rnl 5-bromo-4-chloro-indolylphosphate). All the steps, except the electrotransfer, were performed at room temperature.