Sucrose density gradient isolation

The molar ratio of the components of metaphase chromosome was determined by Uchiyama and his colleagues (Uchiyama et al., 2005). Metaphase chromosomes has been isolated from synchronized human cell line and then purified by sucrose density gradient centrifugation and Percoll density gradient centrifugation. The major components described in this chapter are summarized here. Molar ratio is provided per 100 histone H4 molecules. 

Glycosphingolipid Glycosyltransferase Assays. A 25-33% (vol/vol) homogenate of mouse tumors or harvested cells in 0.32 M sucrose containing 0.1% 2-mer-captoethanol and 0.001 M EDTA (pH 7.0) was used as enzyme source. Membrane fractions for glycolipid gly-cosyltransferase assays were isolated at the junction of 0.32 M and 1.2 M on a discontinuous sucrose density gradient (32,43). 

Subcellular fractionation, sucrose density gradients Allows isolation of specific cellular organelles for organelle proteome analysis difficult to obtain completely pure preparations... 

X-100 in a highly-concentrated phosphate medium to preserve their structural integrity, and then isolated hy differential centrifugation on a sucrose-density gradient. 

Ribosomes needed for translation in the PURE system are isolated from E. coli using sucrose-density gradient centrifugation. The protein factors necessary for translation in E. coli are recombinantly expressed as His-tagged fusions, and purified to homogeneity. These include the factors for initiation (IFl, IF2, and IF3), elongation (EF-G, FF-Tu, FF-Ts), peptide chain release (RFl and RF3), ribosome recycling (RRF), methionyl-tRNA transformylase (MTF) for formylation of the initial Met-tRNA, and the 20 aminoacyl-tRNA synthetases (ARSs) for transfer RNA (tRNA) recy-... 

When an action potential traveling down the axon of a motoneuron reaches the myoneural endplate, a process occurs that releases acetylcholine into the synaptic cleft and consequently depolarizes the postsynaptic membrane. A similar process probably occurs at cholinergic synapses in the central nervous system. In 1950 Fatt and Katz discovered a spontaneous subthreshold activity (MEPP) of motor nerve endings and were thereby led to the concept that acetylcholine is released in definite units (quanta) of 10 to 10 molecules. Electron microscopy subsequently revealed characteristic vesicles about 40 nm in diameter, clustered near presynaptic membranes. Subcellular fractionation procedures were devised by Whittaker and de Robertis for the isolation of these vesicles from brain homogenates in sucrose density gradients, and it was soon demonstrated that they were indeed concentrated reservoirs of acetylcholine. The hypothesis that the vesicles discharge the quanta of transmitter became irresistible. 

An alternative approach to increasing proteomic coverage is based on the analysis of isolated intact mitochondrial protein complexes. One strategy is based on the use of sucrose density gradients to separate intact mitochondrial complexes solubilised with n-dodecyl- 3-D-maltoside (Hanson et al., 2001). Initially the proteins from the individual fractions were analysed by 2-DE. However, subsequently this approach has been coupled with 1-D SDS-PAGE and MALDI PMF analysis of tryptic digests of excised protein bands (Taylor et al., 2003). When applied to human heart mitochondria, this... 

Many properties of informosomes can be studied only after isolation of the particles in a pure state. As we can see, free informosomes of the embryonic cytoplasm resemble in some respects nuclear D-RNPs. They are relatively homogeneous with respect to buoyant density, at least in the case of large informosomes there is a good correlation between the sizes of the informosomes and the RNAs isolated from them. They form a number of discrete components in the sucrose density gradient. All the above-mentioned properties may be explained in terms of a polysomelike structure. However, for such a conclusion it is necessary to have data about the presence of minimal units in informosomes, which data are unavailable at the moment. On the other hand, there are some differences between the nuclear D-RNPs and informosomes with respect to their sedimentation coefficients. The question of the relation between the two types of particles is discussed in detail in the section on The Problem of Messenger RNA Transport (p. 96). 

Fig. 13. Nucleoprotein complexes containing virus-specific RNA in the cytoplasm of HeLa cells infected with vaccina virus. A. Sucrose density gradient centrifugation of cytoplasmic extract, after 30-minute labeling with uridine. Centrifugation 18.5 hours at 22,000 rpm. B. Recentrifugation of RNP isolated from SOS zone sucrose gradient in CsCI density gradient. Centrifugation in preformed CsCI density gradient in SW-39 rotor at 37,000 rpm, 20 hours. (From Belitsina et al. 1968. Molec. BioL (U.S.S.R.), 2 727-735.)...

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