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

The pulse-chased cells were lysed in cold RIPA buffer (10 mM Tris, pH 7.4, I% [wt/vol] Triton X-100, 0.1% [wt/vol] SDS, 0.1% [wt/vol] sodium deoxycholate, 150 mM NaCI) (Tooze et al., 1987) . 20 mM N-ethylmaleimide was added to lysis buffer to prevent spontaneous disulphide bond formation after cell lysis. Lysis was effected by passing the cells through a 21gauge needle several times. The cell extracts and the media were spun for 5 min at 14,000 rpm in an Eppendorf microcentrifuge (Netheler and Hinz GmbH, Hamburg, Germany) to remove the insoluble debris. I00 #1 of RIPA-wasbed Pansorbin was added to each sample to remove proteins that bind to Pansorbin. After overnight rotation at 4~ the Pansorbin was spun out and 5/xl of rabbit antiserum against HBsAg was added to each of the samples, and the mixture was rotated overnight at 4~ 100 #1 of Pansorbin, which had been preincubated overnight with nonradioactive RIPA extract of SV24 cells to block the nonspecific binding sites and washed three times with RIPA, was added to each sample and the samples were rotated overnight at 4"C. The Pansorbin was spun out and the immunoprecipitates were washed three times with cold RIPA and three times with each of the wash buffers used by Tooze et al. (1987) : with cold buffer A (10 mM Tris-HCl, pH 7.4, 0.1% [wt/vol] SDS, 1 mM EDTA), with cold buffer B (10 mM Tris-HC1, pH 7.4, 0.01% [wt/vol] SDS, 1 mM EDTA), with cold buffer C (10 mM Tris-HC1, pH 7.4, 1 mM EDTA, 500 mM NaCI), and finally with cold buffer D (10 mM Tris-HCl, pH 7.4, I mM ElYrA). Between all the washes the immunoprecipitates were spun 3 min at 8,000 rpm in an Eppendorf microcentrifuge.