Sucrose gradients centrifugation

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... 

EC 1.9.3.1]. Purified by selective solubilisation with Triton X-1(X) and subsequently with lauryl maltoside finally by sucrose gradient centrifugation [Li et al. Biochem J 242 417 1978]. 

Carefully load the sample on a sucrose gradient containing heparin and centrifuge as in Section 3.2. 

When discontinuous sucrose gradient centrifugation is used, two different density sucrose solutions should be piled up into the tube. If 20% (w/w) and 30% (w/w) density solutions are piled up, the ER-enriched fraction should be layered on the interphase between 20% (w/w) and 30% (w/w) sucrose solutions. 

A cell extract was separated on a sucrose gradient ranging from 1.1 Op at the top to 1.25p at the bottom and centrifuged to equilibrium. 

If the expression of the mRNA results in a clear signal, a linear sucrose gradient (6-20% sucrose, 5 mM EDTA, 0.25% (w/v) sarcosyl, 15 mM PIPES-NaOH, pH 6.4) is prepared in SW27 tubes or equivalent and the mRNA is carefully loaded on top. After centrifugation (19 h, 80,000 xg), about 33 fractions of 1 ml each are collected, and the mRNA is precipitated using sodium acetate and ethanol. Prior to injection, the RNA of each fraction is pelleted, washed and dissolved in RNAse-free water as described above. Upon injection of 50 ng fractionated mRNA, a two- to fivefold increase in transport activity is expected for the positive mRNA-fraction compared with unfractionated mRNA. All other fractions should not show any specific functional activity. In cases where two neighbouring fractions induce transport activity, pooling of these should be considered. 

DNA methylation induced chromatin fiber compaction only in the presence of bound LH AFM results substantiated by MNase digestion patterns and sucrose gradient centrifugation ... 

ImuVert Manufacturing. The cells were harvested from the fermentation medium and washed by centrifugation, lysed with a French pressure cell and the cell debris was removed by centrifugation. The cell lysis supernatant was then layered on a sucrose gradient and the ImuVert pelleted by centrifugation. The pellet was then resuspended in buffer A (see below) to a nucleic acid concentration of 2 mg/mL. 

Figure 5. Size analysis of Inhibitors I and 11 specific mRNA from levels of 9- and 18-h singly wounded tomato plants and 18-h doubly wounded plants. Poly(A ) RNA was applied to 15-30% linear sucrose gradients and was spun at 25,000 rpm. Twenty-five fractions were collected, the absorbency was measured, and the mRNA was precipitated by cold ethanol. In vitro translations were performed with each fraction in a rabbit reticulocyte system, and isolation of the preinhibitors with preformed antibody precipitates located the position of the two inhibitors. The gradients were calibrated by centrifugation of tomato leaf polyfA)" RNA on an identical gradient. The locations of translatable mRNAs for Inhibitors I and II were identical with RNA obtained from 9- and 18-h singly wounded or 18-h doubly...

Figure 6. Partial purification of Inhibitors I and II mRNA. Fractions containing Inhibitors I and II mRNA determined by in vitro translation analyses were recovered from an initial 15-30% linear sucrose gradient, precipitated by cold ethanol, and applied to a 10-25% linear sucrose gradient. The sample was centrifuged for 36 h at 25,000 rpm. Fractions of the gradient were collected and subjected to in vitro translation analyses. The upper graph represents total methionine incorporation assayed with 1 jiL of the translation mixture as described (ll). The bottom figure quantitates the radiolabel incorporated specifically into Inhibitor I (solid bars) and Inhibitor II (open bars).

In another approach subcellular fractionation of human PMNs, stimulated in vitro with phorbol myristate acetate or opsonized zymsan, was performed by rate-zonal centrifugation in sucrose gradients By varying the centrifugal force the NADPH oxidase could be separated from the activities of enzymes in the lysosomal granules but was similar to the distribution of markers for the plasma membrane, aryl phosphatase, alkaline phosphodiesterase I, and acid aryl phosphatase. 

Selective adsorption on cellulose phosphate CM-cellulose chromatography Centrifugation on sucrose gradient Electrophoresis on sucrose gradient Ultrasonic disintegration DEAE-cellulose chromatography Gel filtration... 

The graph shows E. coli labeled with radioactive thymidine for a short pulse (10 s) followed by a chase with an excess of nonradioactive thymidine. The DNA is then extracted and centrifuged in alkaline sucrose gradients (under high pH conditions the DNA denatures). Explain what these data imply, and interpret these results in light of our current model for DNA replication. 

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