Neutral sucrose gradient

Neutral sucrose gradient sedimentation analysis under high salt conditions [3] shows (Fig. 1) that nucleoids from cells exposed to 100 fjM methotrexate for 1 h sediment more slowly than controls [ratio 0.856 0.04 (SD), n = 3]. If we accept that this reduction in sedimentation rate is due to DNA strand breaks this represents about 300 breaks per genome [4]. 

Fig. 4. DNA strand break assay. DNA strand breaks were monitored by sedimentation of nucleoids through a 15-30% neutral sucrose gradient. Migration of nucleoids is expressed relative to migration of control (undamaged) nucleoids. A 24 h treatment with TG(0) control ( ) +SAB. B 30 min treatment with DMS. C 30 min treatment with DMS + SAB.

Fig. 3 A and B. The weight-average length distribution of duplex AAV DNA molecules observed in the electron microscope has been plotted. A An unfractionated preparation. Circular molecules are shaded. B Fractions representing the major peak in a neutral sucrose gradient were observed. Reprinted by permission of J. Molec. Biol. 79 (1973)...

Fig. 1. Estimation of the size of mRNA for bovine poly(ADP-ribose) synthetase by in vitro translation of size fractionated poly (A) + RNA and Northern blot analysis. A. Size-fractionation of poly (A) + RNA and location of mRNA for the enzyme. Bovine thymus poly(A)+ RNA was size-fractionated by neutral sucrose density gradient centrifugation and RNA in each fraction was translated in vitro. The translated products were immunoprecipitated, and separated on a 7.5% SDS-polyacrylamide gel. B. Typical fluorogram of the gel. Lanes 1, 2, 3, 4, and 5, correspond to fractions 1, 3, 5, 7, and 9, respectively. Molecular weight markers a, p-galactosidase (116K), b, phosphorylase a(95K), c, bovine serum albumin (68K), d, ovalbumin (43K), e, lysozyme (14.3K). C. Northern blot analysis of bovine thymus poly(A)+ RNA. RNA (2 pg per lane) was separated on a 1.2% agarose/formaldehyde gel, transferred to a nitrocellulose filter, and hybridized with the p-iabelled 2.7 kb insert cDNA prepared from the clone ARS-1. Size markers used were 28 S(4.9 kb), 18 S(2.0 kb)rRNA, 3.8 kb, 2.7 kb, and 1.1 kb denatured DNA. Reprinted from Taniguchi etaL, Eur J Biochem 171 571-575,1988.

Gradient-forming materials which provide the densities required for the separation of subcellular particles include salts of alkali metals (e.g. caesium and rubidium chloride), small neutral hydrophilic organic molecules (e.g. sucrose), hydrophilic macromolecules (e.g. proteins and polysaccharides), and a number of miscellaneous compounds more recently introduced and not included in the above group, such as colloidal silica (e.g. Percoll) and non-ionic iodinated aromatic compounds (e.g. Metrizamide, Nycodenz and Renograffin). 

Density gradient stabilization obtained by electrically neutral substances, mostly sucrose. 

The initial steps of solubilization, DEAE chromatography and gel filtration were slight modifications of reported procedures [19]. The pooled fractions from a Sephacryl S-200 (Pharmacia) column were applied to a Mono Q HR5/5 FPLC column (Pharmacia) and eluted in a 20 ml gradient from 0-350 mM NaCl in 0.25 M sucrose-20 mM Tris-HCl pH 7.3. Fractions of 1 ml were collected and assayed for binding of NAA-1-[ C] (61 mCi/mmol, Amersham) by one of three methods [20]. The most active fractions were pooled, desalted and lyophilized. This preparation (approx. 50% receptor) was used either for monoclonal antibody production or was fully purified by native PAGE in a neutral pH discontinuous system [3]. The gel was briefly electroblotted (5 min, 10 mA) to nitrocellulose and the small fraction of transferred proteins visualized by rapid staining [8]. This blot was then used to locate precisely the bulk protein bands remaining in the gel. 

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