Centrifugation In cell organelle suspension or macromolecule solutions exposed to multiples of gravity acceleration (g), the dispersed particles settle to varying degrees depending on their size, shape and density. In centrifuges, this is achieved by rotating a metal rotor with a high-performance motor, in which the centrifuge tubes rotate in a fixed position (angular rotor) or assuming a horizontal position during centrifugation (swing-glass rotor). Acceleration of 600,000 \u00d7 g is also possible in the most powerful ultracentrifuges. Different types of centrifugation can be used for biological purposes.<\/p>\n\n\n\n Differential centrifugation <\/strong><\/p>\n\n\n\n The aim of the method is to isolate and purify the organelles of eukaryotic cells: to fractionate the cells. The examined cells and tissues are first homogenized in an aqueous medium: the cell membrane is destroyed by osmotic means, ultrasound or other mechanical action. The homogenate is then fractionated by repeated centrifugation at increasing speeds: first the nuclei, then the mitochondria, then the vesicles (microsomes) formed from the fragmentation of the endoplasmic reticulum, and finally the free ribosomes. The supernatant obtained by the last centrifugation is the cytosol containing the cell’s solutes.<\/p>\n\n\n\n Hypopycnic gradient centrifugation<\/strong><\/p>\n\n\n\n The method is suitable for the separation of macromolecules and particles of different weights and sedimentation constants. A gradually increasing concentration of sucrose solution is placed in the centrifuge tube towards the bottom of the tube (a continuous gradient is used), the sample to be separated is layered on top of the gradient and then centrifuged until the desired separation is achieved. (The hypopycnic name indicates that the density of the gradient is less than that of the separated particles.) After centrifugation, the bottom of the plastic tube is punctured, the gradient is collected by dripping into tubes and the concentration of test substances in the case of a labelled substance, by radioactivity measurement, etc. The values \u200b\u200bobtained are plotted on a graph. Discontinuous gradient centrifugation is used to prepare high purity preparations of viruses, nuclei, and other cellular components. Two or three solutions of different densities are layered on top of each other, with impurities \u201csticking\u201d at these layer boundaries.<\/p>\n\n\n\n Isopycnic gradient centrifugation <\/strong><\/p>\n\n\n\n A gradient made of a high-density solution (eg caesium chloride) is suitable for the separation of macromolecules and particles according to their density. The sample to be separated is mixed into the caesium chloride solution (Figure 8.4), followed by high-speed ultracentrifugation to form a density gradient in the saline solution, and the sample molecules migrate to the corresponding layer. (Hence the name: isopycnic, i.e., of the same density.) The fractions are collected, the separated substances are detected in the fractions, and the sedimentogram is drawn as described for hypopycnic centrifugation.<\/p>\n<\/span>Types of centrifuges<\/h2>\n<\/span>\n\n\n