STUDIES ON ISOLATED CELL COMPONENTS I. NUCLEAR ISOLATION by DIFFERENTIAL CENTRIFUGATION
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چکیده
THE problem of the isolation of cell components in quantity has been approached in several ways. For the nucleus the methods have included acid-pepsin digestion of the cytoplasm (22), hemolysis of nucleated erythrocytes (1, 33, 19), and recently the differential centrifugation of mechanically disrupted cells. In preparing tissues for differential centrifugation the following procedures have been employed: 1. dessication (5); 2. ultrasonic disintegration (16); S. freezing and thawing (11); 4. heating to 51” C (38); and 5. treatment with citric acid (30, 11, 21, 4). In most of the above methods the tissue is killed before nuclear isolation. Other workers have mechanically broken up living tissue in saline (17, 12) or sucrose solutions (28) and have then isolated the nuclei by differential centrifugation in these solutions. For further references on methods the recent review of Dounce (11) may be consulted. The method of choice for the isolation a,f nuclei will be dictated by the purposes of the experiment, as emphasized by Dounce (11). For the investigation of the physiological and colloid chemical potentialities of the isolated nucleus, which is the aim of the present studies, it becomes necessary to employ procedures which will furnish quantities of pure nuclei in a state resembling the intracellular condition as closely as possible. This means giving attention to such factors as electrolyte composition, tonicity, the maintenance of pH near neutrality during and following extraction, etc. Since technics previously devised for other purposes were not suitable in these respects, modifications have been introduced. A simple method is presented which permits the isolation of rat liver nuclei in a state of purity satisfactory for various colloidal and biochemical studies and in
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تاریخ انتشار 2003