Nuclear extracts sucrose-density-gradient centrifugation

Figure 8 (A) Isolated rat liver nuclear envelope, negatively stained with ammonium molybdate. Note the pronounced electron-transparent octagonal annuli of the nuclear pore complexes. (B) A partly purified nuclear pore complex fraction obtained by Triton X-100 extraction and ultrasonication of nuclear envelope, followed by sucrose density gradient centrifugation. Bars = 200 nm.

To isolate RARs, we routinely incubate cultured cells with radiolabeled retinoic-acid isomers, prepare nuclei, extract the ligand-bound receptors from the purified nuclei and fractionate them using either sucrose density-gradient centrifugation or high-performance size-exclusion chromatography (HPSEC). It is also possible to prepare nuclear extracts from untreated cells and incubate this material with radiolabeled retinoic acid prior to fractionation (3). How-... 

Isopycnic centrifugation. If a post-nuclear extract of hypo-tonically shocked rat TDL is fractionated by means of isopycnic centrifugation in an aqueous sucrose density gradient, all of the sedimentable acid hydrolases, including a small part of cathepsin D, band around a modal density of 1.18 (Fig.l). In agreement with the results presented in Table I, most of the cathepsin D activity is recovered in a soluble, unsedimentable form. The other lysosomal acid hydrolases contribute much less unsedimentable activity. 

Figure 1. Distribution of acid hydrolases and protein after isopycnic centrifugation in sucrose gradients of post-nuclear extracts of rat thoracic duct lymphocytes. The shaded block with a density below 1.10 has an arbitrary density interval of 0.1 and represents the position of the sample layer. The dotted line indicates the histogram obtained if enzyme activity or protein were uniformly distributed. Each histogram shows the average of results with standard deviation, and the number of experiments is given between parentheses.

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... 

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