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1、Virus purification and analysis using OptiPrep-competitive media,SucroseGlycerolCaesium 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 preparati
2、on times and sterilizationCsCl is expensive,CsCl PROBLEMS,Big losses in viral infectivity;low recoveries;high particle:infectivity ratiosSolutions have very high osmolality:viruses lose water;high virus density;high CsCl required for gradientsHighly toxic to cells:must be dialyzed prior to re-infect
3、ion of cellsSolutions 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 iodixanolSolutions are viscous:slow sedimentation of virus particlesHighly toxic to cells:must be dialyzed
4、prior to re-infection of cells,OPTIPREP NO PROBLEMS,Solutions are isoosmotic:virus density is lowSolutions have low viscosity:rapid sedimentation of virus particlesIs non-toxic to cellsHas little or no effect on virus infectivityCells can be re-infected and electrophoresis and HPLC performed without
5、 dialysis,Virus banding density,Effect of medium on PM2 infectivity,Sucrose gradients cause loss of virus surface glycoproteins;no loss with iodixanol gradientsInfectivity recoveries are 10-100 x greater in iodixanol than in sucrose,SUCROSE PROBLEMS(II):for enveloped viruses,particularly retroviruse
6、s,350,000g 1 h,rAAV in discontinuous gradient(V04)Zolotukhin S.et al(1999)Gene Therapy,6,973-985,rAAV in continuous gradient(V04)Hermans,W.T.J.M.C.et al(1999)Human Gene Therapy,10,1885-1891,Gradient Master profiles from 10%and 40%iodixanol at 80 and 20 rpm:effect of time,3 Formats for separation of
7、particles accordingto their density,rAAV in continuous gradient(V04)Hermans,W.T.J.M.C.et al(1999)Human Gene Therapy,10,1885-1891,Comparison of rAAV in CsCl and OptiPrep,Data from Hermans,WTJMC et al(1999)Human GeneTherapy,10,1885-1891,Use of self-generated iodixanol gradients in vertical rotors for
8、virus purification and analysis,Virus purification flow-chart without virus pelleting,Purification of Herpes virus in self-generated 25%iodixanol gradient(V05),Purification of retrovirus:self-generated gradient(V09)Moller-Larsen A&Christensen,T.(1998)J.Virol Meth.,73,151-161,OptiPrepApplication Shee
9、ts for:,AdenovirusEbola virusFoamy virusHerpes simplex virusHIV-1Human papillomavirusLassa virusMurine leukemia virusMurine oncornavirus,Norwalk virusPolyoma virusrAAVRabies virusRous sarcoma virusRetrovirusesSemliki Forest virusSARS Corona virusVaccinia virus,Publications database on viruses,OptiPrep(since 1994)over 600Nycodenz(since 1984)approx 70Using either the Applications CD or the website:www.axis-shield-density-gradient-Follow the instructions to access the relevant IndexClick on the virus of interest,
