Sucrose velocity gradient centrifugation

Chicken embryo fibroblasts (CEFs) are obtained from embryonic day-10 chicken embryos, and cultured as described (Albertinazzi et al, 1998). Transient expression of proteins is achieved by transfection of CEFs by the calcium phosphate technique using 10 fig of plasmid DNA for each 6 cm diameter plate. 18-24 h after transfection, cells are extracted with lysis buffer (1% Triton X-100,150 mM NaCl, 0.5 mg/ml phenyl methyl sulfonyl fluoride (PMSF), 20 mM Tris-Cl, pH 7.5). Cell lysates are clarified by differential centrifugation for 10 min at 18,000g in a refrigerated microcentrifuge, and utilized for analysis by sucrose velocity gradient centrifugation. 

---