The signal sequence of the p62 protein of Semliki Forest virus is involved in initiation but not in completing chain translocation

Total translation mixtures were adjusted to 4% SDS, then boiled for 4 rain and diluted 1:2 with water. 4 vol of immunoprecipitation buffer (2.5 % Triton X-100, 190 mM NaC1, 60 mM Tris-HC1, pH 7.4, 6 mM EDTA, and 20/~g PMSF/rni) and 2 #1 of antibody were added for 16 h at 40C. The mixture was briefly centrifuged (2-3 min in an Eppendorf minifuge) and to the supernatant one fifth volume of a 1:1 slurry of protein A beads were added and incubated at 24"C for 2 h under constant agitation. The beads were collected and washed four times with 1 ml RIPA buffer (Gielkens et al., 1976) by centrifugation, followed by a single wash with a buffer containing 150 mM NaCI, 10 mM Tris-HCl pH 7.4, and 20 t~g PMSF/ml. The beads were then taken up in excess gel loading buffer (Cutler and Garoff, 1986) , heated at 95°C for 5 min, and cleared by centrifugation before loading the immunoprecipitate on the gel.