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

A confluent layer of SV24 cells was labeled with [3~S]methionine overnight with 200 t~Ci/ml in MEM made with dialyzed serum and containing 10% of the normal methionine. The labeling medium was removed and the cells were washed with PBS and lysed. I-IBsAg was immunoprecipitated from aliquots of the iysate, as described above and the washed immunoprecipitates were suspended in 70 #1 of 71.7 mM Na-citrate, pH 5.5, containing 0.143 M 2-mercaptoethanol and 0.15% (wt/vol) SDS, and incubated at 950C for 5 rain. After this mild denaturation to increase oligosaccharide accessibility for the enzyme, the samples were cooled to 37"C. 10/~1 of 1% (wt/vol) BSA was then added, followed by 20 td of endoglycosidase H stock (1 mU//~l). The final incubation buffer contained 50 mM Na-citrate, 0.1 M 2-mereaptoethanol, 0.1% (wt/vol) SDS and 0.2 mU//~l endoglycosidase H. To the control sample, 20 t~l of water was added instead of the enzyme. After 24 h incubation at 37~ the samples were prepared for electrophoresis as described for the pulse-chase experiments, and analyzed by SDS-PAGE.