Density Isopycnic centrifugation

Fig. 4. Localization of WAP27 and WAP20 in the crude microsome fractions and the relation with marker-enzyme activities in three organelles (ER, tonoplast, and Golgi). SDS-PAGE of fractionated proteins by isopycnic linear sucrose density gradient centrifugation of microsome fraction of mulberry cortical parenchyma cells was performed using 6-pL samples in each fraction. Immunoblot analysis was performed with anti-WAP27 and anti-WAP20 antibodies. (From ref. [1], with permission from the American Society of Plant Physiologists.)...

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

Some 37 samples from 16 coals of the Pennsylvania State University coal data base (PSOC) were examined. Separate density fractions were obtained by isopycnic density gradient centrifugation of small ( 3iim) coal particles in an aqueous CsCl density gradient. (4J The individual samples are listed by PSOC numbers, coal description, ASTM designation of coal-rank maceral type, and density in Table I. After separation, the samples... 

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. 

For zonal density gradient centrifugation there are a number of media that can be used, however the most common of these is sucrose whereas the most common medium for isopycnic density gradient centrifugation is caesium chloride (CSCI2). 

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. 

Classical Method. This method (Rl) involves isopycnic centrifugation of cleared lysate in a solution of CsCl containing ethidium bromide (EtBr). EtBr binds by intercalating between DNA base pairs, which causes the DNA to unwind. A covalently closed circular (ccc) DNA molecule such as a plasmid has no free ends and can only unwind to a limited extent, thus limiting the amount of bound EtBr. Linear DNA, such as fragmented chromosomal DNA, has no such topological constraints and can therefore bind more of the EtBr molecules. Because the density of the DNA/EtBr complex decreases as more EtBr is bound, and because more EtBr can be bound to a linear molecule than a covalent circle, the... 

Native apoferritin can be prepared by a number of centrifugal methods. Density gradient centrifugation (62) in isopycnic gradients of CsCl or in sucrose have been used to fractionate native ferritin according to its iron content. The native apoferritin sediments with a buoyant density of 1.23, whilst full ferritin has a density in excess of 1.8. Typically using an SW 27 rotor 100 hr are required at 25.000 rpm with a CsCl... 

The density gradient centrifugation process involves a separation in which the supporting liquid varies in density from the top to the bottom of the tube. Two types are common zonal and isopycnic. Figure 39-21 compares differential, zonal, and isopycnic. 

Isopycnic centrifugation depends solely upon the buoyant density of the particle and not its shape or size and is independent of time. Hence soluble proteins, which have a very similar density (e.g. p = 1.3 g cm 3 in sucrose solution) cannot be usually separated by this method, whereas subcellular organelles (e.g. mitochondria, p = 1.19 g cm, and peroxisomes, p = 1.23 g cm, in sucrose solution) can be effectively separated. 

Fig. 1-3 Isopycnic centrifugation of organeiies. The shading indicates increasing soiu-tion density.

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