Imperforate bowl: centrifugal separators – Process
Reexamination Certificate
2002-01-11
2003-11-04
Cooley, Charles E. (Department: 1723)
Imperforate bowl: centrifugal separators
Process
C220S216000, C141S100000
Reexamination Certificate
active
06641517
ABSTRACT:
BACKGROUND OF THE INVENTION
A. Field of the Invention
The present invention relates to an apparatus and method for making a multiple density layers or gradients of fluid in a vessel in a highly reproducible manner using a float that floats on the surface of the fluid within the vessel.
B. Description of the Related Art
There are various fields where it is desirable to have density layers or gradients of fluid within a vessel for such purposes as the separation of matter, determining density, etc. Such density layers include, for example, a solution retained in a vessel where the fluid is divided into a plurality of layers, each layer having differing concentrations of a soluble material or solute. For example, a bottom or first layer of fluid may have a concentration of a solute that is X moles per liter; a second layer immediately above the first layer may have a concentration of 0.8X moles per liter; a third layer above the second layer may have a concentration of 0.6X moles per liter; and a fourth layer having a concentration of 0.4X moles per liter.
Liquids having gradients of temperature, concentration, density and color have been previously prepared. Liquid density gradients have been used for many years, for a wide variety of purposes, in a number of different industries. The inventor has numerous publications and patents regarding certain aspects of gradient formation and use including Anderson, N. G. Mechanical device for producing density gradients in liquids. Rev. Sci. Instr. 26: 891-892, 1955; Anderson, N. G., Bond, H. E., and Canning, R. E. Analytical techniques for cell fractions. I. Simplified gradient elution programming. Anal. Biochem. 3: 472-478, 1962; Anderson, N. G., and Rutenberg, E. Analytical techniques for cell fractions. A simple gradient-forming apparatus. Anal. Biochem. 21: 259-265, 1967; Candler, E. L., Nunley, C. E., and Anderson, N. G. Analytical techniques for cell fractions. VI. Multiple gradient-distributing rotor (B-XXI). Anal. Biochem. 21: 253-258, 1967.
A variety of other methods for making density gradients have been developed, and Bock, R. M. and Ling, N.-S., Anal. Chem. 26, 1543, 1954, and Morris, C. J. O. R, and Morris, P., Separation Methods in Biochemistry, Pitman Publishing, 2nd ed. (1976) have reviewed many of these. Only one of these methods allowed gradients to be made from multiple solutions, each having a different combination of reagents (Anderson, et al, “Analytical Techniques for Cell Fractions. I. Simplified Gradient Elution Programming”,
Analytical Biochemistry
3: 472-478, 1962) More recent innovations include the use of pumps and pistons, which are differentially controlled by microprocessors, e.g., the Angelique gradient maker (Large Scale Proteomics Corp. Rockville, Md.). Gradients may also be generated during high speed centrifugation by sedimenting a gradient solute such as cesium chloride or an iodinated x-ray contrast medium such as iodixanol. Gradients may be initially prepared as step gradients and linearized by diffusion, by gentle mixing, or by freezing and thawing. A list of references covering existing methods follows.
Density gradients are used to make two basic types of separations. The first separates particles on the basis of sedimentation rate (rate-zonal centrifugation), in which case particles are separated on the basis of the size and density (and to a lesser extent shape) and particles will sediment farther if centrifuged for a longer period of time. The second separates particles on the basis of isopycnic banding density, in which case particles reach their equilibrium density level, and do not sediment farther with continued centrifugation.
Four types of gradients are in general use with either of these basic methods. The first includes step gradients, made by layering a series of solutions of decreasing density (if the solutions are introduced one above the other), and of increasing density (if the solutions are introduced sequentially to the bottom of the tube). The second type comprises linear continuous gradients usually made by a mechanical gradient maker. These are usually introduced slowly through small tubing to the bottom of the centrifuge tube. Linear gradients for either rate zonal or isopycnic zonal centrifugation are useful for resolving very heterogeneous mixtures of particles.
The third type of gradient is non-linear, and may be designed to separate particles having a very wide range of sizes or densities. Non-linear gradient may be designed to separate particles on the basis of both sedimentation rate and isopycnic banding density in the same gradient, in which case some particles reach their isopycnic level at some point in the gradient, while others are still sedimenting. Generally such combined separations involve larger and denser particles which band near the bottom of the gradient, while other smaller, and usually lighter particles are still sedimenting in the upper portion of the gradient.
The fourth type of gradient is generated in a high centrifugal field by sedimentation of the major gradient solute, and is usually used for isopycnic banding.
Many reasons exist for desiring to control gradient shape. Gradient capacity (i.e., the mass of particles which can exist in a zone without causing a density inversion) is a function of gradient slope, and a steep gradient can support a greater mass of particles per unit gradient length than can shallow gradients. The greatest particle mass concentration in a gradient separation usually occurs immediately beneath the sample zone shortly after centrifugation is started. As different particles separate in the length of the gradient, the possibility of an overloaded zone diminishes. For this reason it is desirable to have a short steep gradient section immediately under the sample zone, where the highest gradient capacity is required.
An additional reason for desiring to control gradient shape is that when a population of particles is present that differ little in sedimentation rate, these can best be separated by sedimentation through a longer shallower section of the gradient. Such shallow sections are usually near the center of a gradient.
In the majority of density gradient separations, the gradients and their chemical composition are designed to optimize the separation of one or a few particles types. This accounts for the very large number of different gradient recipes that have been published for subcellular fractionation. Those used for the isolation of mitochondria, for example, are usually quite different from those used to isolate nuclei. For example, traces of divalent cations are required to control nuclear swelling, whereas such ions are generally deleterious to other subcellular particles. Low concentrations of nonionic detergents remove cytoplasmic contamination from nuclei, but are deleterious to the endoplasmic reticulum. Hence there has been no one procedure or gradient that has been optimized for the systematic separation of the majority of all subcellular particles. There is a need for reproducible means for including in gradients zones containing salts, detergents, enzymes and other reactive substances that would increase the number of different subcellular particles separated in one gradient.
Density gradient separations are important in proteomics research. High resolution two-dimensional electrophoresis (2DE) is widely used to produce global maps of the proteins in extracts prepared by solubilizing whole cells or tissues. By careful control of the procedures employed, use of staining procedures which are quantitative, and computerized image analysis and data reduction, quantitative differences in the abundance of individual proteins of ±15% has been achieved (Anderson, N. Leigh, Nance, Sharron L., Tollaksen, Sandra L., Giere, Frederic A., and Anderson, Norman G., Quantitative reproducibility of measurements from Coomassie Blue-stained two-dimensional gels: Analysis of mouse liver protein patterns and a comparison of BALB/c and C57 strains. Electrophoresis 6: 592-599, 1985; Anderson, N. Leigh, Hofmann, Jean
Cooley Charles E.
Davis Garrett V.
Large Scale Proteomics Corporation
Robbins John C.
Sorkin David
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