病毒纯化与分析 文档资料.ppt

Virus purification and analysis using OptiPrep ? - competitive media ? Sucrose ? Glycerol ? Caesium chloride SOLUTION HANDLING ? Density solutions prepared by diluting OptiPrep ? with culture medium or any balanced salt solution (Application Sheet V01) ? Sterile solution ? CsCl/sucrose solutions require lengthy preparation times and sterilization ? CsCl is expensive CsCl PROBLEMS ? Big losses in viral infectivity; low recoveries; high particle:infectivity ratios ? Solutions have very high osmolality: viruses lose water; high virus density; high [CsCl] required for gradients ? Highly toxic to cells: must be dialyzed prior to re-infection of cells ? Solutions ionic and corrosive: samples must be dialyzed before electrophoresis or HPLC SUCROSE PROBLEMS (I) ? Solutions are hyperosmotic: virus density in sucrose usually higher than in iodixanol ? Solutions are viscous: slow sedimentation of virus particles ? Highly toxic to cells: must be dialyzed prior to re-infection of cells OPTIPREP – NO PROBLEMS ? Solutions are isoosmotic: virus density is low ? Solutions have low viscosity: rapid sedimentation of virus particles ? Is non-toxic to cells ? Has little or no effect on virus infectivity ? Cells can be re-infected and electrophoresis and HPLC performed without dialysis Virus banding density Virus CsCl Sucrose Iodixanol Epstein-Barr 1.26 1.14 Semliki Forest 1.26 1.18 1.18 Poliovirus 1.34 1.26 1.22 Measles 1.21 1.18 1.16 Kivela et al (1999)Virol. 262, 364-374 0 10 20 30 40 50 60 % iodixanol, sucrose, glycerol 10 11 10 12 1.1 1.3 1.5 1.7 CsCl g/ml Iodixanol Glycerol CsCl Sucrose I n f e c t i v i t y Effect of medium on PM2 infectivity ? Sucrose gradients cause loss of virus surface glycoproteins; no loss with iodixanol gradients ? Infectivity recoveries are 10-100x greater in iodixanol than in sucrose SUCROSE PROBLEMS (II): for enveloped viruses, particularly ret