Первый слайд презентации: L. 3 DIFFERENTIAL CENTRIFUGATION
Principles of centrifugation Centrifugation Through Density Gradients Subcellular Fractionation and Marker Enzymes
Cell are disrupted in a homogenizer and the resulting mixture, called the homogenate. The broken cell preparation is referred to as cell-free preparation or ‘homogenate’. Properly prepared, a homogenate is the sum of all the cel components obtained by rupturing the cell membrane. Organelles and other subcellular components can be isolated by differential centrifugation. Differential centrifugation is carried out by centrifuging a sample at low speed and separating the supernatant and pellet. The supernatant is then recentrifuged at higher speed and the supernatant and pellet separated again The denser ( более плотный ) material forms a pellet at lower centrifugal force than will the less-denser material. The isolated fractions is used for further purification.
The centrifuge is primarily used to separated biological components based upon differential sedimentation properties. Many types of centrifuges are available for various applications. All centrifuges basically consist of a motor which spins a rotor containing the experimental sample. The differences between centrifuges are in the speeds at which the samples are centrifuged and the volumes of samples.
The centrifugal force exerted on a particle in the solution is expressed in multiples of the force exerted by gravity. The centrifugal force is proportional to the radius of the centrifugal head and to the square of angular velocity. Hence, it is more convenient to use relatively small heads rotating at high speeds. Thus, a head of approximately 10 cm diameter rotating at about 40,000 rpm will produce nearly 100,000 g. At such high speeds, the head is run in a vacuum to prevent the heat produced by air friction. The tube containing the homogenate is usually held at an angle to the axis of rotating to keep the path of particles through the solution as short as possible
Слайд 10: Separation of cell organelles by differential centrifugation
Field Time Structure(s) separated 700 g 10 min Nuclei, cell membranes 5,000 g 10 min Mitochondria, lysosomes 57,000 g 60 min Microsomes, lysosomes 150,000 g 30 min Ribosomes
Слайд 11: A Summary of Centrifuge Techniques and Applications
Preparative Velocity sedimentation centrifugation (pelleting) Separation and isolation of particles in a solution. May be applied to precipitates, cell organelles, cells, or biomolecules. Fractional centrifugation Isolation of particles, based on size, by successive centrifugation at increasing rotor speeds.
Слайд 12: Centrifugation Through Density Gradients
Fast sedimenting particles will be contaminated with slow sedimenting particles. The reason for this is that by the time large particles near the top of the tube are pelleted, some of the small particles near the bottom of the tube have also pelleted. In addition, mechanical vibrations, thermal gradients and convection currents ( конвекционные потоки ) can also affect the sedimentation properties. Centrifugation through a dense medium, or density gradient centrifugation, can alleviate these problems. In addition, density gradient centrifugation allow for better separation of particles with similar properties.
Several different media are commonly used in density gradient centrifugation depending upon the exact application Sucrose • CsCl • Ficoll • Hypaque • Percoll
Слайд 14: Types of density gradient centrifugation
The two types of density gradient centrifugation are rate zonal and isopycnic ( or equilibrium ). In rate zonal centrifugation the density of the particles being separated are greater than the density of the solvent. Separation is based primarily upon size (i.e., larger particles will sediment faster ). It is important to determine the optimal length of centrifugation for separating the particle of interest. If the centrifuge is not turned off soon enough all of particles will pellet. In isopycnic centrifugation the solvent density encompasses density of particles. The separation is based upon particle density. Centrifugation is carried out until equilibrium is reached (i.e., all particles have banded at densities corresponding to their own). In practice, a mixture of fractions is obtained on single centrifugation. In order to obtain pure fractions, the precipitates have to be recentrifuged many times.
Слайд 15: Density gradient centrifugation
Zonal Gradient is present in the tube before centrifugation and sample is layered on top. Used to isolate purified molecules and determine s. Isopycnic Gradient is formed during centrifugation. Used to isolate purified molecules and determine s.
Density gradients can be preformed or formed during centrifugation. In the case of isopycnic density gradients it is possible to mix the sample with the desity gradient medium and carry out the centrifugation until the density gradient forms. The various components in the sample will then be found at a position which corresponds to their density. This is especially useful in the case of Percoll which rapidly forms density gradients when subjected to centrifugation. Another common example in which self-forming gradients are commonly used is the separation of nucleic acids on CsCl gradients (Nucleic Acid Isolation ).
Слайд 18: Subcellular Fractionation and Marker Enzyme
Organelle Marker nuclei DNA mitochondria cytochrome oxidase lysosome hydrolases peroxisome catalase Golgi α- mannosidase plasma memb rane adenylate cyclase cytos ol lactate ehydrogenase