An accurate slicer of disc acrylamide gels devised by simple modification of a density gradient fractionator.

A commercially available density gradient fractionator has been modified in a simple and inexpensive way to yield a device for accurately slicing disc acrylamide gels. Attractive features of this device include convenience of operation and the capacity to easily vary thickness of the gel slice.     

Evaluation of density gradient separation methods

A new method for evaluating the effectiveness of isooynic separation processes is developed and explored. This technique is designed to measure the ability of a separation technique to attain the goals of a sorting process. The performance of a separation process is expressed in terms of an inconsistency number, I, whose values ranges from zero to unity. Processes which function perfectly are characterized by an I value of zero. Separation processes which subdivide a sample are characterized by an I value of unity. The validity of using I to measure the performance of separation processes was established by demonstrating that alterations in the performance of a well characterized separation technique were fully reflected by changes in the I value for the process. The capacity of four different techniques for ordering red cells with respect to their density was then shown to lie in a definite hierarchy. This hierarchy can be expressed as: Murphy technique (I = 0.21) > albumin density gradient (I = 0.43) > Stractan density gradient I = 0.53) > Percoll density gradient (I = 0.73). The performance of these techniques was found to be dependent on the exact operating conditions employed and the specific fraction of red cells isolated.     

Purification of very high density lipoproteins by differential density gradient ultracentrifugation

Differential density gradient ultracentrifugation procedures, utilizing a vertical rotor, were developed for the preparative purification of very high density lipoproteins (VHDL, density greater than 1.21 g/ml). The VHDLs of several insect species were purified as follows. An initial density gradient ultracentrifugation step removed lipoproteins of lower density from the VHDL-fraction, which partially separated from the nonlipoproteins present in the infranatant. A complete separation was achieved by a second centrifugation step employing a modified gradient system. The use of a vertical rotor and specially designed discontinuous gradients allows a relatively fast, efficient, and economical isolation of the class of very high density lipoproteins. Similar gradient systems should be useful for the detection and purification of VHDLs from other sources.     

Kinetics of sedimentation in a density gradient

Two models of sedimentation in a density gradient are analyzed. The first is for sedimentation in cylindrical sector geometry and contains the assumption that diffusion can be neglected. The second treats sedimentation in a rectangular field and includes diffusion, although the boundaries are not treated exactly. In both of these models we approximate the time dependence of the gradient by a relaxation form. We derive exact results for both models. It is also shown that the sedimentation coefficient can be calculated from data by following the motion of the position of the maximum (or minimum) of the concentration gradient.     

Computational analysis of density gradient distribution profiles

A method is presented for the construction of distribution profiles following subcellular fractionation by density gradient centrifugation. The method allows reconstruction of the frequency-density distributions over user-defined density limits, enabling averaging of results from multiple experiments. An alternative format for data presentation, that of relative concentration versus density, is also introduced. This overcomes some practical problems associated with frequency-density distribution profiles. The technique utilizes a direct noniterative approach to curve fitting and can easily be implemented on microcomputers.     

Enucleation of cells by density gradient centrifugation

HEp-2 cells can be enucleated by ultracentrifugation in a colloidal silica (PTL) density gradient, containing cytochalasin B. Under optimal conditions, more than 70% of the cells are enucleated. Purification up to 97% is carried out by centrifugation at low speed through a second, preformed PTL density gradient. The enucleated cells show a high viability, as tested by [³H]leucine incorporation. The method described was developed for enucleation of high quantities of cells and has the advantage that it can be used for cell types which do not adhere firmly enough to a carrier to be centrifuged as a monolayer.     

Physico-chemical properties of low density lipoproteins isolated in an equilibrium density gradient

The tryptophanyls of total low density lipoproteins (LDL) (1.006-1.063 g/ml) from coronary heart disease (CHD) patients and subjects without CHD signs had different accessibility to fluorescence quenchers (I-and acrylamide). LDL were separated into subfractions in equilibrium density gradient. The coefficient of extinction , quantum yield and other spectral characteristics of LDL intrinsic fluorescence, rotational mobility of maleimide spin labels and fatty acid spin probe were different in LDL subfractions from healthy subjects. LDL subfractions with hydrated density 1.045-1.05 g/ml bound to B,E-receptors of cultured fibroblasts more effectively than did subfractions with density 1.01-1.03 g/ml. Structural differences of apo-B in the particles with different lipid to protein ratio are supposed.     

Rapid density gradient centrifugation using Short Column Techniques

The Short Column Technique is a useful technique for rate zonal and isopycnic density gradient runs with both the swinging bucket and fixed angle rotors, We have described and illustrated its advantages of significant time saving and the elimination of the need for tube caps. Thick-walled polycarbonate tubes are used. While some loss in resolution may result with some samples using short columns. It is our opinlon that the technique can be widely adopted to provide useful information.     

Separation of hormone-sensitive yeast cells by density gradient centrifugation

Cells in cultures of haploid strains of Saccharomyces cerevisiaein stationary phase were separated into interface fraction andpellet fraction by density gradient centrifugation. Cells inpellet fraction expanded in response to yeast sexual hormoneand animal sex hormones, whereas cells in interface fractiondid not. ¹Present address: Department of Biology, Faculty of Science,Osaka City University, Sumiyoshi-ku, Osaka, Japan (Received July 16, 1970; )