Centrifugation isopycnic

The isopycnic method has been used to dramatically demonstrate semiconservative DNA replication, using CsCl density gradients. Separation of DNA, RNA, protein, and carbohydrates can be performed in dense CsCl solutions, where the RNA pellets, the DNA forms bands, and protein and carbohydrates form a thin layer called a pellicle at the top of the gradient. 

If self-forming cesium salt gradients are used, take care to be within the solubility of the salt at the given temperature to avoid crystallizations during centrifugation, since salt crystals may destroy the rotor during run (solubilities of cesium chloride and sulfate are given in Table 5.4). 

The maximal speed of a rotor has to be reduced if solutions with densities p 1.2g/ml are used. The proper revolutions per minute Nre j are calculated by 

1 Purification of High Moiecuiar Weight DMA in CsCi Gradients 

This protocol is an example of separation using a self-generating density gradient. 

Dissolve 2.0 g cesium chloride (ultrapure) in 2.0 ml of Soln. A, containing 5-8 pg DNA. Stir with a glass rod at 20 °C until the salt is completely dissolved. Pour the solution into a centrifuge 

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Fig. 2 SDS-polyacrylamide gel electrophoresis of T. vaginalis hydrogenosomes purified by isopycnic centrifugation on a Percoll gradient. 1 PFOR 2 malic enzyme, 64-kDa hy-drogenase, and Cpn 60 3 succinyl CoA synthetase fi subunit 4 Hmp35 5 succinyl CoA synthetase a subunit 6 Hmp31 (ATP/ADP carrier) 7 adenylate kinase 8 thiol peroxidase. Well-resolved but unmarked bands mostly belong to malic enzyme fragments or unknown proteins. Proteins were identified by mass spectroscopy. The 12% gel is stained with Coomassie Brilliant Blue R 250 (authors original)...

C Isopycnic centrifugation the density gradient forms during centrifugation. Illustration courtesy of Beckman Instruments, Inc. 

Fig. 1-2 Isopycnic centrifugation of organelles. The shading indicates increasing solution density.

Mitochondria are about the size of bacteria. They have a diameter of 0.2 to 0.5 gm and are 0.5 to 7 p.m long. They are bounded by two lipid bilayers, the inner one being highly folded. These folds are called cristae. The innermost space of the mitochondrion is called the matrix. They have their own DNA in the form of at least one copy of a circular double helix (Chap. 7), about 5 p.m in overall diameter it differs from nuclear DNA in its density and denaturation temperature by virtue of being richer in guanosine and cytosine (Chap. 7). The different density from nuclear DNA allows its separation by isopycnic centrifugation. Mitochondria also have their own type of ribosomes that differ from those in the cytoplasm but are similar to those of bacteria. 

Figure 6.8 Diagrammatic illustration of zonal and isopycnic centrifugation.

Figure 9-16. Protein distribution observed after isopycnic centrifugation of crude particles from castor bean endosperm on linear A) and stepped (B) sucrose gradients. The upper curves of each panel show the measured sucrose concentration in each fraction and the lower curves, the protein. [From T. G. Cooper and H. Beevers, J. Biol. Chem., 244 3507 (1969).]...

The methods covered in detail in this manual are mainly those of chromatography and electrophoresis. Although isopycnic centrifugation is covered ( 11.3), sedimentation velocity methods are only touched on ( 11.2). It is intended to cover these in a future manual. The other major omission is the use of cellulose acetate and DEAE-... 

The density, p, of a double stranded DNA molecule, and hence its position in the gradient, depends primarily on its nucleotide composition (eq. 2.2 gives p= 1.660+0.098 (GC)). The relation does not hold for DNAs containing glucosylated, methylated or other modified residues, nor for DNAs of very simple sequence such as synthetic polynucleotides, crab poly dAT (Wells et al. 1970) and centromeric DNA. Single stranded DNA is denser than double stranded DNA and isopycnic centrifugation can be used to separate them. 

If apoptosis is only occurring at relatively low levels in cultures, it may be necessary to obtain a purified population of apoptotic cells prior to DNA isolation and electrophoresis. This may be achieved by exploiting the fact that apoptotic cells are more dense than normal cells. Hence it is possible to purify apoptotic cells by isopycnic centrifugation. Percoll can be used to create solutions of different densities. The precise Percoll densities used for isolation of apoptotic cells will depend on the cell type under investigation. 

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