Density gradient centrifugations

As an example for differential centrifugation, this protocol gives the preparation of membranes of the sarcoplasmic Ontracellular) reticulum (SR) of heart muscle. 

Centrifuge the combined supernatants with 100 000 x g for 45 min (e.g., Beckman Coulter Type 45 Ti rotor 35 000 rpm). 

Discard the supernatant and resuspend the precipitated SR vesicles in total 1 - 2 ml of Soln. C, freeze quickly in liquid nitrogen and store aliquots at -70 °C. 

Density gradients maybe divided into three types preformed discontinuous gradients (e.g.. Protocol 5.3.1), preformed continuous gradients (e.g.. Protocol 5.3.2) or self-generating gradients (e.g.. Protocol 5.3.3). Materials used for density gradients are classified as ionic and non-ionic media (Table 5.1). 

Gradients made by a distinct substance are not only characterized by the density, but also by viscosity and ionic strength and osmolarity (Table 5.2). Since gradient solutions of high density mostly are concentrated solutions, it should be kept in mind 

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Illustration showing separation by equilibrium-density-gradient centrifugation. The homogeneous mixture in (a) separates into three bands (b) after applying centrifugal force. 

Heavy isotopes endow the compounds in which they appear with slightly greater masses than their unlabeled counterparts. These compounds can be separated and quantitated by mass spectrometry (or density gradient centrifugation, if they are macromolecules). For example, O was used in separate experiments as a tracer of the fate of the oxygen atoms in water and carbon dioxide to determine whether the atmospheric oxygen produced in photosynthesis arose from HgO, COg, or both ... 

The purification is done by sucrose density-gradient centrifugation (DeSa and Hastings, 1968 Hastings and Dunlap, 1986). Six sucrose... 

The PemB cellular localisation was determined both in E. chrysanthenu and in an E. coli recombinant strain by Western blot of the cell fractions with a PemB-antiserum. No PemB was detected in the culture supernatant and only trace amounts were found in the soluble cell fractions - periplasm and cytoplasm (Figure 2). PemB was found mostly in the total membrane fraction from which it could be completely extracted by Triton X-100/Mg2+ and partially extracted by Sarkosyl (Figure 2). This behaviour is typical of inner membrane proteins, but since some exceptions have been noticed it does not positively indicate the PemB localisation (15). We performed cell membrane fractionation in sucrose density gradient centrifugation both by sedimentation and flotation, using several markers of inner and outer membrane vesicles. PemB was found in the outer membrane vesicles (data not shown). 

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.

Fig. 4. Localization of WAP27 and WAP20 in the crude microsome fractions and the relation with marker-enzyme activities in three organelles (ER, tonoplast, and Golgi). SDS-PAGE of fractionated proteins by isopycnic linear sucrose density gradient centrifugation of microsome fraction of mulberry cortical parenchyma cells was performed using 6-pL samples in each fraction. Immunoblot analysis was performed with anti-WAP27 and anti-WAP20 antibodies. (From ref. [1], with permission from the American Society of Plant Physiologists.)...

Figure 13.7 Caesium chloride density gradient centrifugation for (a) the separation of DNA from RNA and protein and (b) the separation of linear DNA and supercoiled DNA.

Ion-exchange HPLC can also be useful in the separation of larger nucleic acid molecules. One such application is as an alternative to CsCl density gradient centrifugation in the preparation of plasmids. Plasmid molecules typically consist of between 1000 and 10 000 base pairs. The plasmid is first isolated from the bacterial cell by alkaline lysis and pure plasmid obtained from this crude extract by a one-step chromatographic separation. 

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