Density gradient centrifugation sucrose gradients, preparation

The microsome fractions see Fig. 1) that were prepared from mulberry cortical parenchyma cells were fractionated to 24 or 25 fractions using the 15-50% sucrose linear density gradient centrifugation see Fig. 2). Profiles of the marker enzymes and the protein content are described in Fig. 3. In general, the antimycin A-insensitive cytochrome c reductase activity is exhibited at a lower density than are those of the marker enzymes. The fraction that exhibited the highest antimycin A-insensitive cytochrome c reductase activity for each month was used as the ER-enriched fraction. 

Fig. 2. Preparation of linear sucrose density gradient, centrifugation, and fractionation. Fractionation is performed using an ISCO model 640 density gradient fractionator in this experiment.

Following such treatment, the cultured cells in monolayer are washed with phosphate-buffered saline and extracted in 4 ml 0.5% Triton X-100 in saline/EDTA (100 mM NaCl, 10 mM EDTA, pH 8.0) for 2 min at room temperature. This releases most of the cytoplasmic material whilst the nuclei remain attached to the culture dish. 0.5% sodium dodecyl sulphate and 40 //g/ml pancreatic RNAse (preincubated at 80°C for 10 min, to inactivate DNAse) in saline/EDTA (above) is then added and the mixture incubated for 20 min at 37°C. One volume of chloroform/isoamyl alcohol (20 5, v/v) is then added and the phases mixed gently. The aqueous phase is separated by centrifugation and extracted again with chloroform/isoamyl alcohol. DNA is precipitated from the aqueous phase with 2 vol 95% ethanol and resuspended in 0.01 M Tris, pH 7.5. Alkaline sucrose density gradients (5-20%) are prepared in 0.1 M NaCl, 0.1 M NaOH with a final volume of 4.1 ml. Samples of DNA (max 3 fig) are layered on the top of these gradients and spun at 32 000 rpm at 20°C in a SW.50.1 Beckman rotor for 120 min. Fractions are collected and the [3H]DNA precipitated... 

In 1980, Mullet, Burke and Arntzen attempted to prepare the so-called native PS-I complex from pea thylakoids (see Chapter 26). The fraction representing photosystem I obtained by sucrose density-gradient centrifugation was found to contain both chlorophylls a and b and have a chlorophyll-to-P700 ratio of 110 10 and a Chi alb ratio 18. Based on the pigment-to-P700 ratio, the authors named their preparation PSI-110. Further fractionation of PSI-110 resulted in a subfraction called PSI-65 that contained... 

Density gradient centrifugation in 10-40% (w/v) sucrose in isotonic saline was an early method to separate IgG and IgM (McCall and Potter, 1973). This method is still used for the preparation of chicken IgM, for 16-18 h at 10°C at 35000 rpm (Beckman SW-39 rotor). IgM (19S) sediments close to the bottom but dilution may be significant. 

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 principal problem in preparation of mitochondrial DNA is to avoid contamination by nuclear DNA One strategr is to prepare a highly purified fi-action of mitochondria, using cellular firactionation (differential centrifugation, sucrose gradients). Another is to prepare a total extract of DNA (or an extract solely enriched in mtDNA) and to separate nuclear and mitochondrial DNA taking advantage of their differential physical properties (physical conformation, density). The last possibility is to work with the mixture of DNAs from the total extract and to use a probe, complementary of the mtDNA... 

Fig. 1. Profiles of pigment distribution after rate-zone sedimentation. Cell-free extracts were layered onto 5-35% (wl/wt) sucrose gradients prepared over a 4 ml cushion of 60% sucrose and centrifuged at 4 C for 240 min in a Beckman SW27 rotor at 27,000 rpm. The density and antenna BChl concentrations were determined from the refractive index and deconvolution of the absorption spectrum (16) respectively.

Moore (1974) examined PG synthesis in castor bean endosperm. Sucrose density gradient centrifugation showed equal amounts of activity in the ER and mitochondrial fractions. The two preparations were remarkably similar, having a pH optimum of 7.3 and an optimal Triton X-100 concentration of 0.075% Mn was superior to Mg in both cases, with optimal concentrations of 5 mM and 1-2 mM, respectively. The for glycerol-3-P was 50 fiM, and for CDP-DG 2 /aM. The mitochondrial preparation showed greater dependence on added CDP-DG. Moore (1974) reported inhibition by reagents that react with SH groups but did not note whether PGP accumulated. In the standard assay at least 90% of the product was PG. 

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