Density gradient fractionation by piston displacement.

A method for the fractionation of centrifuged density gradients is described. A piston forced into the centrifuge tube from above displaces the gradient, while its tip collects liquid from the small volume immediately below it without disturbing the unfractionated part of the gradient. The centrifuge tube holder permits sensitive visual detection of bands of light scattering particles which may then be rapidly and precisely extracted. Continuous fractionation of the entire gradient is also possible.     

Density gradient system: II. A 50 channel programmable undulating diphragm peristaltic pump

A special peristaltic pump is described that has functioned as part of a system for density gradient formation and fractionation. Twenty-five pumping tubes are actuated on both the top and bottom of the pump. A Mylar diaphragm interposed between the rollers and the pumping tubes filters out the horizontal, stretching component of the forces imparted to the tubes. This greatly prolongs tube life, increases the allowable pressures that can be achieved with such a pump, and thus permits accurate delivery of viscous solutions.An explanation of the cause of the pulsations produced by peristaltic pumps is presented and the virtual elimination of these pulsations is demonstrated.Both velocity and direction of flow of the pump are controlled. By means of two independent bidirectional digital counters, preset volumes of fluid can be delivered and the total volume of liquid determined.Studies demonstrating the relative independence of fluid volume delivered at a preset count versus flow velocity and composition are presented.Other possibilities for use of the pump in automating density gradient analysis are discussed. Possibilities for employment of the pump for autoanalysis and in artificial organs are indicated.     

Simultation of gradient and band propagation in the centrifuge.

A technique is developed for simulating the behavior of both the gradient-forming solute and macromolecular bands in a centrifuge. The change with time of the density gradient due to diffusion and sedimentation of the gradient-forming solute is calculated by a finite difference method, making use of the results of earlier work on the theory of the equilibrium density gradient. Using a perturbation technique, the concentration profiles of dilute bands of macromolecules are then calculated as they sediment and diffuse through the varying supporting gradient. Results of the stimulaion techniques are compared with experiment.     

Multiple fraction collection using a peristaltic pump and a fraction collector modified for multiple channel operation

The fractionation of gradients in sedimentation analysis of proteins is a time-consuming operation. This operation can be performed more rapidly by using a multichannel peristaltic pump and two or more fraction collectors. Specifically designed fraction collectors are available for multiple fraction collection, notably the Slave Micro-Fractionator (Model SFC-80)¹ from Gilson Medical Electronics, Inc. Use of multiple fraction collectors during the fractionation of gradients has the disadvantage that these units occupy considerable space and are expensive. In addition, the total number of fractions collected from one gradient is often considerably less than the capacity of most fraction collectors. To offset the space and expense disadvantages, we have devised a modification for the Gilson Micro-Fractionator (Model FC-80H)¹ which permits three sets of 25 fractions each to be collected simultaneously from 3 gradients using only 1 fraction collector. A multichannel peristaltic pump is employed, and an attachment which permits three drop tubes to be positioned above the rack of fraction tubes is secured to the drop detector head¹ of the Micro-Fractionator. One drop tube is clamped in the normal manner in the drop detector head, and fraction size is determined by counting drops in the normal manner. Fractions from the other two drop tubes are not counted. Alternatively, fraction size can be determined by adjusting the pump speed and using the timer on the fraction collector.     

An apparatus for density gradient forming and nonpuncturing fractionation

A technique is described which allows gradient formation, sample layering, and nonpuncturing gradient fractionation in one apparatus. The demonstrated device can be adapted to centrifuge tubes of different sizes and volumes and connected to almost any device which is used for gradient formation or analysis with flow cuvettes and/or fraction collectors. The operation technique can be standardized. The construction gives reproducible gradients with sharp starting bands of the samples and provides good resolution after centrifugation.     

Separation of human metaphase chromosomes at neutral pH by velocity sedimentation at 20 times gravity

A method for the separation of large quantities of human metaphase chromosomes at neutral pH is described. The separation was performed in a specially designed sedimentation chamber which was placed in a bucket of a speed-controlled centrifuge. Flow deflectors in the chamber allowed both the undisturbed introduction of gradient and chromosomes, as well as the undisturbed fractionation of the content of the chamber. The centrifuge was run at 20 g for 1 h taking care of slow acceleration and deceleration. The slow morphological changes of the chromosomes due to ageing at neutral pH do not affect the resolving power of the sedimentation technique within this hour. This in contrast to separation of chromosomes at neutral pH by velocity sedimentation at unit gravity during 20 h, as described before. The main advantage of chromosome sorting at neutral pH is that the fractionated chromosomes are more suitable for gene transfer and gene mapping experiments.     

A semiautomated system for the production and analysis of sucrose density gradients

A semiautomated system permitting considerable accuracy, speed and reproducibility in the making and fractionation of sucrose density gradients is described. The system consists of a modified Beckman gradient forming device which makes six gradients simultaneously and delivers them into six 12.5 ml polyallomer centrifuge tubes in such a manner that new material is continuously added to the meniscus of the gradient. The gradients are fractionated three at a time and up to 100 fractions per gradient can be collected automatically directly into scintillation vials with a choice of drop counting or time mode with rinse and automatic addition of scintillation fluid to each vial. The system can process up to six gradients per hour but centrifugation time is usually the limiting factor. With neutral sucrose gradients, sharp, reproducible, monodisperse peaks containing up to 100% of the gradient radioactivity are usually obtained but a smaller monodisperse peak containing as little as 3.5% of the gradient radioactivity can be detected under conditions where some pairs of molecules might tangle or dimerize. The resolution and reproducibility of this system when used with neutral sucrose gradients is at least the equal if not superior to that commonly claimed for alkaline sucrose gradients.     

A separation chamber to sort cells,nuclei, and chromosomes at moderate g forces. II. Studies on velocity sedimentation and equilibrium density centrifugation of mammalian cells

A separation chamber having a surface of 50 cm² and a height of 2 cm is described for the rapid separation of cells and cell organelles at acceleration forces from 10 to 90g. To eliminate wall sedimentation artifacts, the chamber was positioned 20 cm from the rotor axis in a speed-controlled centrifuge. The chamber has flow deflectors for the undisturbed introduction of the sample layer and the gradient; an antivortex cross prevents swirling upon acceleration and deceleration. To illustrate the use of the separation chamber, examples of velocity sedimentation and of equilibrium density centrifugation are given: (i) human monocytes (70% were 90% pure) are separated from lymphocytes in 10 min at 20g; (ii) nonparenchymal rat liver cells are separated in 10 min at 16g in 97% pure endothelial cells and 99% pure Kupffer cells; (iii) equilibrium density centrifugation of human peripheral blood cells at about 90g permits the separation of erythrocytes, monocytes, lymphocytes, neutrophils, eosinophils, and basophils in one run. B cells are separated from T cells. The movement of swinging buckets is analyzed in mathematical terms and a simple method is offered to determine the position of cells in density gradients with the use of a small programmable calculator.     

An ultramicro method for quantitation of amino acids in biological fluids

A modification of the commercially available SZ-14 reorienting density gradient rotor is described whereby continuous sample flow with density gradient isopycnic banding may be utilized. This permits the fractionation of large volumes of dilute homogenates with excellent recovery and purity. The technique is demonstrated for the isolation of nuclei and particles of mitochondrial size.     

A device for a discontinuous fractionation of liquid density gradients yielding high resolution

When a liquid density gradient is fractionated through the bottom of a tube turbulences occur near the hole resulting in a mixing of closely spaced bands. To prevent this mixing the gradient is pumped upward and, in conventional instruments, guided through tubings into a fraction collector. Since mixing of bands in the tubings may occur, the advantage of pumping the gradient upward is diminished. To prevent this mixing an apparatus is described which, with the help of a peristaltic pump, collects fractions discontinuously after the gradient has been pumped upward thus preventing a mixing in instruments used for analysis and tubings attached to the fractionator.     

A New Procedure for Quantitative Measurements of Virus Particles in Crude Preparations

A new method is described for the quantitative measurement of virus concentration in crude preparations by density gradient centrifugation and electron microscopy. The centrifugation is carried out in a specially designed centrifuge tube which permits separation and sedimentation of virus particles at different levels according to their sedimentation velocity. The gradient of a mixture of heavy and normal water (D(2)O-H(2)O) is designed to sediment the virus particles with constant velocity so that the optimal time of centrifugation can easily be calculated. The virus particles are collected on carbon-coated nickel grids floating on mercury at the bottom of the centrifuge tube and are counted by means of electron microscopy. The efficiency of the method is demonstrated with a crude plant extract of tobacco mosaic virus.     

Technique and apparatus for automated fractionation of the contents of small centrifuge tubes: application to analytical ultracentrifugation

An automated method is described for dividing the contents of small cylindrical centrifuge tubes into fractions deriving from laminae of solution as thin as 0.1 mm in the direction of the cylindrical axis. Experimental data are presented to demonstrate that fractions as small as 1 microliter may be prepared with a standard deviation of less than 3% in volume delivery and that negligible mixing occurs between the contents of adjacent fractions during the fractionation procedure. The method has been used to quantitate the gradients of a variety of radiolabeled proteins formed in sedimentation velocity and sedimentation equilibrium experiments. Sedimentation coefficients and molecular weights calculated from the gradients obtained agree well with literature values and with values obtained by optically scanning the centrifuge tubes (A.K. Attri and A.P. Minton, 1983, Anal. Biochem. 133, 142-152; 1984, Anal. Biochem. 136, 407-415). The present technique combines a spatial resolution equal to that of optical methods of gradient measurement with a sensitivity which may be several orders of magnitude greater, depending upon the specific activity of labeled solute.     

Rapid screening for plasmid DNA

Summary A procedure is described for demonstrating plasmid DNA and its molecular weight, based on rate zonal centrifugation of unlabelled DNA in neutral sucrose gradients containing a low concentration of ethidium bromide. Each DNA species is then visualized as a discrete fluorescent band when the centrifuge tube is illuminated with ultra-violet light. Plasmids exist as closed circular and as relaxed circular molecules, which sediment separately, but during preparation of lysates, closed circular molecules are nicked so that each plasmid forms only a single band of relaxed circles within the gradient.     

A sterile, regulated, pulsation-dampened precision dosing system for biomedical technics

For sterile continuous metering of small and very small flow rates delivered by peristaltic pump, a gravimetric control system which permits a high accuracy of mass flow is described. This is achieved by a digital PI controller and a digital filter with which the very accurate signal from a high precision electronic scale is filtered. In addition proposals are made for solving the problem of refilling the reservoir and for compensating the pulsations caused by the peristaltic pump.     

A new unit gravity sedimentation chamber

A unit gravity sedimentation chamber with a flow deflector of special design is described. The flow deflector consists of a rotational-symmetric Perspex body fitted in the top cone of the chamber. This deflector allows both the undisturbed introduction of a buffered step gradient and the undisturbed application of an extremely thin sample layer (0.28 mm) as well as the undisturbed fractionation of the contents of the chamber. The operational quality of the method is substantiated by the separation of : (i) murine leukemia G₁,S- and G₂,M-phase cells; (ii) human lymphocytes from monocytes; (iii) diploid and tetraploid rat liver nuclei; and (iv) isolated chromosomes from Chinese hamster into different size classes.     

Solubilization and fractionation of rat liver chromatin

Rat liver chromatin can be dissolved in dilute aqueous solutions of salyrgan to give a concentrated chromatin solution suitable for fractionation by sedimentation or density gradient centrifugation.     

Purification of the protein crystal from Bacillus thuringiensis by zonal gradient centrifugation.

A method is described for the large-scale purification of the Bacillus thuringiensis protein crystal by zonal gradient centrifugation. NaBr gradients are employed in a Beckman J21-B centrifuge equipped with a JCF-Z rotor.     

Isoelectric focusing of cells using zwitterionic buffers.

A simple method of isoelectric focusing of cells is described. The pH gradient, superimposed on a density gradient, is developed by generating opposing concentration gradients of two zwitterionic buffers. The method can be used as a cell separation technique or as a means of characterizing the cell type on the basis of the focusing pH. Focusing is rapid and thus the method is of special advantage in its application to cells.     

The generation of non-linear solute gradients for chromatography by using only simple apparatus.

It is shown that by using only a series of tubes with constant cross-sectional area and a single-channel peristaltic pump, it is possible to produce solute gradients with time profiles that are concave, convex, sigmoid or even with turning points. The general theory for predicting gradients, by using tubes in series that are either open or closed to the atmosphere, is presented, and the equations using three compartments are given. Experiments are described that support the usefulness and accuracy of such a theoretical treatment.     

Fractionation of Different Life Cycle Stages of Microsporidia Nosema grylli from Crickets Gryllus bimaculatus by Centrifugation in Percoll Density Gradient for Biochemical Research

ABSTRACT. A new method of fractionation and purification of different life cycle stages of microsporidia Nosema grylli , parasitizing the fat body of cricket Gryllus bimaculatus , by centrifugation in Percoll density gradient is elaborated. The whole procedure can be summarized as: 1) infected fat body preparation, 2) homogenization in buffer and filtration through cotton wad and filter paper, 3) first centrifuging, resulting in the separation of the pellet into three layers containing different life cycle stages, 4) second centrifuging of the chosen layer in Percoll density gradient, 5) washing out the Percoll from the fraction under study. After centrifugation in Percoll density gradient, meronts and early sporonts form a band in the area corresponding to density 1.016 g/ml. Mature spores form the pellet at the bottom of centrifuge tube, while immature spores are distributed throughout the layer of 1.016 g/ml up to the bottom of the centrifuge tube, according to their buoyant densities. The offered technique is simple, it takes about one hour and may become a routine procedure for biochemical studies on microsporidia.