Gradient fractionation, sucrose

Southern hybridization experiments, employing the cloned PHA synthase structural gene of Acinetobacter sp. and sucrose gradient fractions of DNA preparations separated in plasmid and chromosomal DNA fractions gave two hybridization signals and revealed some but not yet conclusive evidence for... 

Example polysome profiles from sucrose gradient fractionation of HeLa cell lysates, either untreated or treated with EDTA, are shown in Fig. 6.4A and B. The polysome profile of untreated HeLa lysates shows three defined peaks in less dense fractions (6 to 9), which correspond to the 80S, 60S, and 40S peaks (Fig. 6.4A). Treatment of lysates with 30 /iM EDTA results in ribosome dissociation leaving predominantly free 60S and 40S subunits... 

A technical challenge with this step is to achieve RNA extraction of uniform quality and efficiency for each fraction. This is because the amount of RNA in each sucrose gradient fraction varies considerably and the high concentration of sucrose in the bottom fractions interferes with phase separation in typical phenol-based extraction steps. To address these problems, we spike each fraction with an aliquot of a foreign (control) RNA, which can be used later to correct for differences in RNA recovery (and reverse transcription efficiency) between samples. We then remove sucrose from the samples by precipitation of total nucleic acid and protein with ethanol. To purify RNA, a standard Trizol (Invitrogen) extraction is performed as outlined later (also see product insert). 

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.

The sequence of steps in the biosynthesis of the ferritin iron core has been studied by analyzing the incorporation of Fe into ferritin during synthesis of the protein vivo. Ferritin, collected at various intervals after the induction of synthesis, was fractionated according to iron core size by sedimentation through gradients of sucrose (32). Fe appeared first in ferritin with small amounts of Fe, and later, the Fe appeared in fractions further down the gradient as the core size and the ratio... 

Subcellular fractionation, sucrose density gradients Allows isolation of specific cellular organelles for organelle proteome analysis difficult to obtain completely pure preparations... 

Fig. 5.9. Slot-blot analysis of GAPDH mRNA from a sucrose gradient fractionation. Upper panel UV26o profiles of a cytoplasmic lysate in a 20-47% (w/w) sucrose gradient containing either 5mM Mgz+ (solid curve), or 10 mM EDTA (dotted curve). Lower panel Autoradiograph from the slot-blot analysis of fractions from both gradients hybridised with a probe specific for glyceraldehyde 3-phosphate dehydrogenase. Notice the altered sedimentation behaviour of GAPDH mRNA as a result of EDTA-mediated release from ribosomes.

The presence of NaCl in buffers at concentrations of 50-300 m A/ was examined in order to ascertain if increased ionic strength may improve release of chromatin-associated proteins prior to the sucrose step gradient. Low salt concentrations (50-150 mM) did not improve histone release, and high salt concentrations (200-300 mM) reduced the yield of nucleoli and increased the amount of Noplp detected in upper step gradient fractions. 

FIGURE 1. A) Polypeptide composition of PSII membranes (1) and PSII complex (2). B) Sucrose gradient fractionation of PSII complex. 

For the isolation of psliG2, Hindlll digested total DNA was separated on a 10 -30 % sucrose gradient. Fractions enriched in fragment sizes of 5 - 6 kb were subcloned in Bluescript KS. About 700 colonies were screened by Southern blot analysis with a psbG specific probe under low stingency conditions and six positive clones were identified. 

Figure 1 Products of LS mRNA translation in the presence of 1 /il gradient fractions obtained by ultracentrifugation of chloroplast extract through a sucrose gradient (10 - 30%, SW50, 4 hours 50000 rpm). 1,2) fractions 20, 28 in 5 jul translation mix without added RNA 3-12) fractions 1f3,4,5,6,8,12,20,28, each in 5 pi translation mix containing 25 pg/ml LS mRNA.

Addition of poly(l) poly(c) to the control extract did not modify the rate of degradation of ( H) mengo ENA, but in the interferon extract hydrolysis was 2-fold higher after only 5 niin (Figure 6). By 50 min virtually no material remained in the cold acid precipitable fraction. Sucrose density gradient analysis showed that addition of poly(l) poly(c) to the interferon-treated cell extract led to the complete disappearance of the 34 S peak and the appearance of most of the radioactivity at the top of the gradient. Almost identical results were obtained when the nuclease... 

Table 1. Distribution of EMC replicase, protein and lipid P in the isopycnic sucrose gradient fractions ...

The particle size limit is estimated by equation (4) however, this equation does not give the particle size distribution within the floating chylomicron fraction. Pinter and Zilversmit (1962) and Zilversmit (1963) have described an important and readily applied method for estimating the particle size distribution. They employed ultracentrifugal-flotation in concentrated sucrose gradients. Linear sucrose gradients were prepared so that density and viscosity at a point within the gradient were described by the equations ... 

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