Gradient Fractionation

Fraction Enzyme Activity (ftmoles substrate utilized/g (fresh wt)/hr)  

Both these fractionation methods permit the contents of the centrifuge tube to drip sequentially into a set of test tubes. Ideally it would be desirable for each test tube or fraction to receive the same volume of solution. Unfortunately this is often not the case. Droplets obtained from the least dense region of the gradient are also the least viscous, and therefore exhibit a smaller amount of surface tension than do droplets obtained from denser regions. Since the size a droplet can attain before falling away from the tube is a function of its surface tension, the drops obtained from the top of the gradient are smaller than those obtained from the bottom. If quantitation of the gradient contents is desired, this problem must be eliminated or appropriate corrections made for it. 

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It has been established that methane is produced on rice roots by reduction of CO2. This was examined in rice roots using a combination of 16S rRNA sequencing and density gradient fractionation of C-labeled DNA after incubation with C02. The major groups of archaea detected were Methanosarcinaceae that decreased with time to be replaced by the hitherto uncultured Rice Cluster I, although the former subsequently dominated (Lu et al. 2005). 

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... 

Automated gradient fractionator with continuous UV (260 nm) flow cell (Teledyne Isco Lincoln, NE)... 

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). 

Fractionate the gradient solution into 1.2 mL aliquots by a gradient fractionator (ISCO, Model 640). 

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.

Fractions of 1 CV or less may be collected across the gradient. Fractions from the main product peak and from each side can be analyzed by SDS-PAGE with silver stain. Contaminants can then be identified by their molecular weights. Extrapolating the eluting salt concentration at peak center for each component of interest will allow you to map their relative retention characteristics as a function of both pH and salt concentration. 

Figure 7. HPLC analysis of a mutagenic drinking water concentrate (neutral fraction). On a Bondapak C/s column, 15 X 20 pL of a drinking water concentrate (12.5 X ltf-fold concentrated) was separated by using a H2O-C2H3N gradient. Fractions were pooled as indicated. After reconcentration, the fractions were assayed for mutagenic activity in the Salmonella...

In this approach, residues from a sample extract were concentrated on a column in pure phosphate buffer and then eluted with a MeCN gradient. Fractions corresponding to each analyte of interest (1.5-ml fraction centered on the retention time) were collected and analyzed under different chromatographic conditions. 

Garson, M. J., Flowers, A. E., Webb, R. I., Charan, R. D., and McCaffrey, E. J., A sponge/dinoflagellate association in the haplosclerid sponge Haliclona sp. cellular origin of cytotoxic alkaloids by Percoll density gradient fractionation, Cell Tissue Res., 293, 365, 1998. 

Ion-exchange chromatography DEAE-Sephadex elution via pH gradient. Fractions 50-60, 5 ml each, pooled, dialyzed, and concentrated 25 9 225 88 9.8 2200 44 23.6... 

Ion exchange chromatography DEAE-Sephadex elution via KC gradient. Fractions 21-31, 2 ml each, pooled and concentrated 5 1 35 364 52 1820 36.4 125... 

Hydroxyl apatite chromatography elution via phosphate buffer gradient. Fractions 15-18, 1 ml each. f / r 1 CnA on lonn... 

The density gradient fractionator marketed by MSE provides a useful stand for these methods. The hollow Allen screw adaptor can be fitted to the top mount and the bottom mount can be used as... 

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