Sucrose density

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). 

Sphaeroplasts were prepared by slight modifications to published methods [12,13]. Lysis of sphaeroplasts was effected by a combination of osmotic lysis and gentle mechanical disruption [14]. Discontinuous sucrose-density gradients were constructed and fractions were then assayed for protein, PG and marker enzymes for different organelles. 

The subcellular location of PG was studied in cells disrupted by osmotic lysis through formation and disruption of sphaeroplasts from self-induced anaerobically-grown cells. A discontinuous sucrose-density gradient produced four bands labelled I, II, III and IV. Band I included many vesicles and a peak of alkaline phosphatase activity (a vacuolar marker in yeasts), NADPH cytochrome c oxidoreductase activity, an endoplasmic reticulum marker, and... 

Before use, thaw the required number of gradient tubes overnight at 4°, to establish continuous sucrose density gradients. 

August to June. Because the density of the ER differs with the season, we collected ER by the linear sucrose density gradient method. 

Overlay 4 mL of crude microsomal fraction onto the linear sucrose density gradient solution. 

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. 3. Distribution ofvarious membrane markers (ER, tonoplast, Golgi complex, and mitochondrion) in the fractions of linear sucrose density gradient fractionation of mulberry cortical parenchyma cells in February.

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.)...

The molar ratio of the components of metaphase chromosome was determined by Uchiyama and his colleagues (Uchiyama et al., 2005). Metaphase chromosomes has been isolated from synchronized human cell line and then purified by sucrose density gradient centrifugation and Percoll density gradient centrifugation. The major components described in this chapter are summarized here. Molar ratio is provided per 100 histone H4 molecules. 

Samples of gallbladder bile obtained in this way were analysed for bile acids, phospholipids and cholesterol (from which the cholesterol saturation indices were derived). Biliary bile-acid composition was then measured by HPLC. The vesicles were separated from micelles by sucrose density gradient ultra-centrifugation and the cholesterol microcrystal nucleation time measured as described above. 

RNA Separation by Non-Denaturating Sucrose Density Gradient Centrifugation... 

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