Percoll密度梯度离心教程课件.pptVIP

Percoll密度梯度离心教程;Outline;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 assays;Principles of density gradient centrifugation;Separation by density (isopycnic centrifugation);Percoll – physical properties;Storage of Percoll;Sterilization of Percoll solutions;How to make and use gradients of Percoll—Making and diluting a stock solution of Percoll;How to make and use gradients of Percoll—Diluting stock solutions to lower densities;Tips of making and use gradients of Percoll ;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 20°C. (swinging bucket rotor ) 3. Approximately 2 × 109 cells (200 OD600) were pelleted, resuspended in 1 ml Tris buffer, overlaid onto the preformed gradient, and centrifuged at 400 gav for 60 min.;Percoll gradient purification of spores;Instrument and operation;Instrument and operation;Instrument and operation;Instrument and operation—data export;References and applications; contrast