Step density gradient

Two types of gradients may be prepared discontinuous or step density gradient, or continuous density gradient. 

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. 

Method Density gradient. Rate-zonal. The rate-zonal method is one of six addressed by SpinPro. The other methods are differential, differential-flotation, discontinuous, isopycnic, and 2-step isopycnic. These methods differ dramatically in their set up, principles of operation, and expected results. The rate-zonal method is described here briefly so that the recommendations to follow can be appreciated. Prior to the run in a rate-zonal method, a gradient material is introduced to the rotor tubes in steps of increasing density from the top to the bottom of the tube. The sample to be separated is layered, as a thin band, on the top of the gradient. As the run begins, each component in the sample moves toward the bottom of the tube. Some components sediment faster than others. This fact is the basis for the separation. If the run parameters are appropriate, the components will form separate bands within the gradient. At the conclusion of the run, the band representing the component of interest can be removed from the tube. 

Preparation of Glucan Synthase Fractions. Microsomal and plasma membranes were isolated by differential and density-gradient centrifugation. CHAPS-solubilized glucan synthase (CSGS) was prepared by the two-step procedure (4,9). In step 1, contaminating proteins were extracted with... 

The duration of the potential step (the time interval i of Es) is usually determined by the type of information that the experimenter desires of the particular system. The time may vary from as little as 10 /is to as long as several seconds. The minimum time is limited by the ability of the potentiostat to charge the electrode, whereas the maximum time is determined by the onset of convection from vibrations or density gradients. Special shielded electrodes enable times on the order of 100 s to be reached with no apparent disturbance of the diffiisional process. 

Enrichment of rare cancer cells from peripheral blood samples is an application that typically requires density gradient centrifugation as a first step. Application of this technique addresses two objectives depletion of erythrocytes and depletion of polymorphonuclear cells. It is expected that cancer cells undergo sedimentation with the mononuclear cell fraction because of their similar density. However, some studies have found that cancer cells are also lost in the polymorphonuclear fraction or the erythrocyte fraction (8,9). Optimization of the density gradient sedimentation step is an important issue in such an application, because it will determine the recovery of rare cells from blood and affect the chances of their detection by immunochemical means. 

The following section describes the steps necessary to isolate carcinoma cells (cell line, MCF-7) from a buffy coat. A filtration step is added at the end of the density gradient centrifugation procedure to concentrate cells onto a polycarbonate membrane, which is a convenient vehicle for analysis by immunocytochemistry. 

Density is one of the properties that may be used to separate minerals. There are many procedures for obtaining so-called heavy mineral fractions from soils. These fractions consist of minerals with densities greater than those of the more common minerals such as quartz and feldspars. In a previous section the classical use of density gradients to compare soils was mentioned. A logical further step is the precise identification of the various minerals in the different fractions, particularly the denser fractions, since these are likely to be the most diagnostic. 

Whitesides and coworkers combined microfluidic networks with a PDMS platform to create patterned gradients of biomolecules on a surface.121 This method involves a two-step process (1) formation of a gradient of avidin within well-defined patterns by use of microfluidic channels and (2) specific interaction between the avidin gradient pattern and biotin. Such patterns with a density gradient of immobilized biomolecules may find application in studies on cell development and function. 

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