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1、Percoll density gradient centrifugation,Outline,Principles Physical properties Storage Make and use density of Percoll Tips Protocol Instrument and operation References and applications Contrast(differential velocity centrifugation),For biological particles, the ideal gradient medium has been descri
2、bed 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
3、 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 assays,Principles of density gradient ce
4、ntrifugation,The equation for the sedimentation of a sphere in a centrifugal field is:V= 2 ( p l ) 18 where v = sedimentation rated = diameter of the particle (hydrodynamically equivalent sphere)p= particle density l= liquid density = viscosity of the mediumg = centrifugal force,Separation by densit
5、y (isopycnic centrifugation),Separation by size (rate zonal centrifugation),Percoll physical properties,Percoll 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/ml Conductivity 1.0 mS/cm（电导率） Osmolali
6、ty 25 mOsm/kg H2O（渗透压） Viscosity 10 5 cP at 20C（粘度） pH 9.0 0.5 at 20C Refractive Index 1.3540 0.005 at 20C（折射率） Percoll is non-toxic,1.01.3g/ml,The viscosity of Percoll is lower in saline solutions at physiological ionic,Storage of Percoll,Sterile and unopen-5 years When opened, Percoll should be st
7、ored 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 the gradient is sterile and is not physically disturbed If stored at -18C, gradients form upon thawing
8、, necessitating a mixing of the contents of the bottle before use.,Sterilization of Percoll solutions,120C for 30 min Absence of salts or sucrose. Minimum contact with air (narrow-necked bottle) If Percoll form particles, these particles may be removed by low speed centrifugation. If any significant
9、 evaporation occurs during autoclaving, the volume should be replenished with sterile water so that the density is not affected.,How to make and use gradients of Percoll Making and diluting a stock solution of Percoll,Where Vx = volume of diluting medium (ml)Vo = volume of undiluted Percoll (ml)o =
10、density of Percoll (1.130 + 0.005 g/ml*)10 = density of 1.5 M NaCl = 1.058 g/mldensity of 2.5 M sucrose = 1.316 g/mli= density of SIP solution produced (g/ml) Thus, for SIP in saline, i = 1.123 g/ml,for SIP in sucrose, i = 1.149 g/ml, assuming o = 1.130 g/ml.,Adding 9 parts (v/v) of Percoll to 1 par
11、t (v/v) of 1.5 M NaCl or 10 concentrated cell culture medium is a simple way of preparing a Stock Isotonic Percoll (SIP) solution.,“渗透活性物质的物质的量除以溶液的体积称为溶液的渗透浓度（osmolarity），单位为mol/L或mmol/L”,How to make and use gradients of Percoll Diluting stock solutions to lower densities,Solutions of Stock Isotoni
12、c Percoll (SIP) are diluted to lower densities simply by adding 0.15 M NaCl (or normal strength cell culture medium) for cell work, or with 0.25 M sucrose when working with subcellular particles or viruses.,Tips of making and use gradients of Percoll,The colloid does not perceptibly diffuse over tim
13、e, resulting in the formation of very stable gradients. Therefore, both discontinuous and continuous gradients can be prepared weeks in advance, giving great reproducibility over the course of an experiment. Measure the weight of the solution, make sure the weight of solution are all the same.,实验重复性
14、的保证,1. Percoll was diluted 9:1 (vol/vol) with 1.5 M NaCl, 2. Put 10 ml of the Percoll solution into 15-ml Corex tubes and centrifuged at 19,240 gav for 15 min at 20C. (swinging bucket rotor ) 3. Approximately 2 109 cells (200 OD600) were pelleted, resuspended in 1 ml Tris buffer, overlaid onto the p
15、reformed gradient, and centrifuged at 400 gav for 60 min.,Isolation of quiescent and nonquiescent cells form yeast stationary-phase culture,Allen C. The Journal of cell biology .2006,Percoll gradient purification of spores,Strains were grown overnight in YPD liquid medium. Cells were washed twice wi
16、th ddH2O and diluted to a final cell density of OD600 = 0.5. Then, 10 l of equal-volume mixed cells were spotted on V8 medium and incubated for seven days in the dark at room temperature. The entire mating patch was suspended in 60% Percoll (GE Health) in PBS with 0.1% Triton X100. After centrifugat
17、ion at 10,000 X g for 30 mins in an SW41Ti ultracentrifuge rotor (Beckman-Coulter), a band of spores near the bottom of the Percoll gradient was recovered with a 1-ml tuberculin syringe and transferred into an Eppendorf tube. The total spore production was determined by multiplying the spore density, measured by hemocytometer, with the final volume.,
