Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2003-02-26
2004-09-21
Nguyen, Nam (Department: 1753)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S600000, C204S644000, C204S459000, C204S610000
Reexamination Certificate
active
06793791
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is related in general to electrophoretic apparatus and procedures for the separation and purification of biological materials. In particular, it pertains to a device for gel-free isoelectric focusing of fluids in relatively small and variable volumes.
2. Description of the Related Art
U.S. Pat. No. 4,897,169 (1990) taught that fluid stabilization can also be achieved by rapid recycling of process fluid through narrow channels formed between parallel flat plates. Another approach to fluid stabilization, disclosed in U.S. Pat. No. 4,588,492, consists of rotating the separation assembly around its horizontal axis. A commercial implementation of this concept, marketed by BIO-RAD Laboratories of Hercules, Calif., under the trademark ROTOFOR, received significant acceptance for scientific applications.
Nevertheless, there remained a need for instruments requiring smaller priming volumes, because available research quantities of protein solutions are often very small. A significant step toward that end was achieved by the invention described in U.S. Pat. No. 5,540,826 (1996). This disclosure taught that the multiple compartments of the instrument's cell need not be arranged in parallel, in filter-press like assemblies, but can be aligned in serpentine fashion, resembling somewhat cross-flow filtration. The patent taught streamlining of fluid flow using a narrow passage between adjacent cavities, or using a septum, such as a monofilament screen. In practice, though, implementation of these concepts proved to be functional but complicated (see Baygents et al., “Recycling Electrophoretic Separations: Modeling of Isotachophoresis and Isoelectric Focusing,” J. Chromatogr. A, 779:165-183, 1997).
Therefore, there is still a need for simple devices that will separate by isoelectric focusing variable volumes of priming solution, say in the range of 0.5 to 5 ml. The present invention provides an easy and practical approach to accomplish these objectives while retaining the advantages achieved with prior-art instruments.
BRIEF SUMMARY OF THE INVENTION
In view of the foregoing, the present invention is intended to provide a device of simple construction for practicing isoelectric focusing of free solutions in variable volumes in the range of about 0.5-5 ml, an impossible task with prior art equipment. In addition, the invention is directed at a cell configuration that facilitates priming and collection of separated fractions without inter-compartmental mixing while retaining an effective cooling of the process fluid.
Therefore, according to one aspect of the invention, a deformable elastomer tube section, filled to the desired volume with free solution, is used as the focusing cell of an IEF unit. The tube is provided with a leak-proof inlet port for priming the cell with IEF fluid and with end electrode compartments or coupling means to connect the tube to external electrodes. One longitudinal side of the cell is placed in contact with a substantially horizontal cold plate that supports the cell and provides a heat sink to dissipate Joule heating generated by the electric field. The opposite longitudinal side of the cell is engaged by a plurality of transverse parallel rods impinging upon and increasingly pressing against the flexible surface of the cell. This compression causes a swelling of the skin of the cell between each pair of rods and forms distinct contiguous fluid-filled bubble-compartments for IEF separations. Fluid stabilization is achieved by means of control of the depth of gaps between contiguous bubbles. As isoelectric focusing progresses in conventional manner, the gap between the rods and the plate is progressively reduced in order to create separate contiguous fluid compartments which, at the end of the process, contain discrete products of separation. The plate, the rods and the deformable cell are assembled in a structure capable of rotation along the cell's horizontal axis to minimize gravitational convection within said compartments.
In the preferred procedure, at the assumed end of the IEF process the gaps between contiguous compartments in the focusing cell are closed completely by squeezing the transverse rods against the support plate. Thus, each bubble-compartment becomes isolated from adjacent ones. The contents of the bubbles can now be collected simultaneously or individually by puncturing the skin by means of hollow collection needles attached to syringes or tubing sections. A series of collection needles aligned with the compartments of the focusing cell can be used to simultaneously collect the contents of all compartments at the end of the focusing procedure.
According to yet another aspect of the invention, the focusing cell is manufactured with an expandable material, such as natural or synthetic rubber or silicone rubber, so that the total volume of free solution subjected to the IEF process may be varied and controlled by the priming step. In addition, cells of various diameters and length may be used with the same device to provide alternative ranges of volumes for IEF separation. The number of rods in the compression grid can be varied at will. Most likely, the grid will contain not less than 4 and not more than 19 rods, thus forming between 5 and 20 bubbles, respectively.
Various other purposes and advantages of the invention will become clear from its description in the specification that follows and from the novel features particularly pointed out in the appended claims. Therefore, for the accomplishment of the objectives described above, this invention consists of the features hereinafter illustrated in the drawings, fully described in the detailed description of the preferred embodiment and particularly pointed out in the claims. However, such drawings and description disclose but one of the various ways in which the invention may be practiced.
REFERENCES:
patent: 3616456 (1971-10-01), Valmet
patent: 3915839 (1975-10-01), Rilbe et al.
patent: 4234404 (1980-11-01), Satoh
patent: 4401538 (1983-08-01), Hausfeld
patent: 4588492 (1986-05-01), Bier
patent: 4897169 (1990-01-01), Bier et al.
patent: 5173164 (1992-12-01), Egen et al.
patent: 5540826 (1996-07-01), Bier et al.
Baygents et al., “Recycling Electrophoretic Separations: Modeling of Isotachophoresis and Isoelectric Focusing,” J. Chromatgr. A, 779:165-183, 1997.
Durando Antonio R.
Mutschler Brian L
Nguyen Nam
Quarles & Brady Streich & Lang LLP
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