Percoll-密度梯度离心教程.pptxVIP

Percoll density gradient centrifugationOutlinePrinciplesPhysical propertiesStorageMake and use density of PercollTipsProtocolInstrument and operationReferences and applicationsContrast(differential velocity centrifugation)For biological particles, the ideal gradient medium has been described as one having the following characteristics:? covers a sufficient density range for isopycnic banding of all biological particles of interest? possesses physiological ionic strength and pH? is iso-osmotic throughout the gradient? has low viscosity? is non-toxic? will not penetrate biological membranes? is supplied sterile and is resterilizable? will form self-generated gradients by centrifugation at moderate g-forces? is compatible with biological materials? is easily removed from purified materials? does not affect assay procedures? will not quench radioactive assaysPrinciples of density gradient centrifugation?The equation for the sedimentation of a sphere in a centrifugal field is: V=where v = sedimentation rate d = diameter of the particle (hydrodynamically equivalent sphere) ρp= particle density ρl= liquid density η = viscosity of the medium g = centrifugal forceSeparation by density(isopycnic centrifugation)Separation by size (rate zonal centrifugation)Percoll – physical propertiesPercoll is available from GE Healthcare.Composition silica sol with nondialyzable polyvinylpyrrolidone (PVP) coating（由聚乙烯吡咯烷酮包裹的硅胶颗粒）Density 1.130 ± 0.005 g/mlConductivity 1.0 mS/cm（电导率）Osmolality 25 mOsm/kg H2O（渗透压）Viscosity 10 ± 5 cP at 20°C（粘度）pH 9.0 ± 0.5 at 20°CRefractive Index 1.3540 ± 0.005 at 20°C（折射率）Percoll is non-toxic1.0~1.3g/ml The viscosity of Percoll is lower in saline solutions atphysiological ionicStorage of PercollSterile and unopen----5 yearsWhen opened, Percoll should be stored below +8℃If Percoll opened under non-sterile conditions, it can be frozen for up to 6minths at -18℃Preformed gradients can be stored for weeks without a change in gradient shape, provided that