Density gradient centrifugation separation

Figure 1. Procedure for density gradient centrifugation separation of coal macerals.

Fig. 6.1. (a) CsCl density gradient centrifugation separates DNA from RNA and proteins due to their different buoyant densities, (b) By addition of ethidium bromide, chromosomal DNA can be separated from plasmid DNA. 

Illustration showing separation by equilibrium-density-gradient centrifugation. The homogeneous mixture in (a) separates into three bands (b) after applying centrifugal force. 

Heavy isotopes endow the compounds in which they appear with slightly greater masses than their unlabeled counterparts. These compounds can be separated and quantitated by mass spectrometry (or density gradient centrifugation, if they are macromolecules). For example, O was used in separate experiments as a tracer of the fate of the oxygen atoms in water and carbon dioxide to determine whether the atmospheric oxygen produced in photosynthesis arose from HgO, COg, or both ... 

Figure 13.7 Caesium chloride density gradient centrifugation for (a) the separation of DNA from RNA and protein and (b) the separation of linear DNA and supercoiled DNA.

Ion-exchange HPLC can also be useful in the separation of larger nucleic acid molecules. One such application is as an alternative to CsCl density gradient centrifugation in the preparation of plasmids. Plasmid molecules typically consist of between 1000 and 10 000 base pairs. The plasmid is first isolated from the bacterial cell by alkaline lysis and pure plasmid obtained from this crude extract by a one-step chromatographic separation. 

Density gradient centrifugation is used to separate macromolecules that differ only slightly in size or density. Two techniques are commonly used. 

PF had been proposed as the terminal complex (23) and associated pores were reported on the outer membrane EF (24). Due to their proximity to the site of cellulose ribbon extrusion from the cell surface, these structures were assumed to be responsible for cellulose synthesis. A model was advanced in which cellulose synthase was localized on the outer membrane, which invoked adhesion sites between the outer and plasma membranes as a mechanism to explain the transfer of uridine-diphosphoryl-glucose (UDPG) from the cytoplasm to the cellulose synthases (25,26). However, when the outer and plasma membranes of Acetobacter were isolated separately by density-gradient centrifugation, the cellulose synthase activity was localized only in the plasma membrane fraction (27). Therefore, the linear structures observed on the Acetobacter outer membrane, while they may be associated in some manner with cellulose biosynthesis, are probably not the cellulose synthase terminal complexes. Since no ultrastructural evidence for adhesion sites between the outer and plasma membranes has been presented, a thorough investigation of the mechanism of / (1-4) glucan chain translocation from the cytoplasmic membrane to the outer membrane in Acetobacter xylinvm is now in order. 

RNA Separation by Non-Denaturating Sucrose Density Gradient Centrifugation... 

Figure 12.2 Separation of DNA samples by density-gradient centrifugation. Peak 1 is the DNA molecule containing 14 N, while peak 2 contains 15 N. Reprinted by permission from I. Tinoco, Jr., K. Sauer, and J. C. Wang, Physical Chemistry. Principles and Applications in Biological Science, Prentice Hall, Upper Saddle River, New Jersey, 1995, p. 292.

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