Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
1999-11-23
2001-04-10
Ludlow, Jan (Department: 1743)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
C204S606000, C204S616000
Reexamination Certificate
active
06214191
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and apparatus for electrophoretically separating and characterizing sample analytes. In particular, the invention relates to multidimensional electrophoresis.
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BACKGROUND OF THE INVENTION
For decades, electrophoretic separation methods have been central to identifying and characterizing chemical and biochemical samples. In the usual procedure, an electrophoresis tube or slab is filled with a fluid electrophoresis medium, and the fluid medium is covalently cross-linked or temperature-solidified to form a non-flowable, stabilized gel separation medium. A sample is loaded into one end of the tube, or into one or more wells of the slab gel, and an electric field is generated to draw the samples through the medium. Electrophoretic separation may depend predominantly on molecular size, e.g., in the cases of nucleic acids and SDS-bathed proteins, or on a combination of size and charge, as in the case of non-denaturing gel electrophoresis of polypeptides or polysaccharides, for example.
Isoelectric focusing (IEF) is an electrophoresis method based on the migration of a molecular species in a pH gradient to its isoelectric point (pI). The pH gradient is established by subjecting an ampholyte solution containing a large number of different-pI species to an electric field, usually in a crosslinked matrix. Analytes added to the equilibrated ampholyte-containing medium will migrate to their isoelectric points along the pH gradient.
For complex samples, multidimensional electrophoresis methods have been employed to better separate species that comigrate when only a single electrophoresis dimension is used. The conventional approach to two dimensional electrophoresis is to perform the first dimension in a rigid, usually crosslinked matrix. For analysis of proteins, for example, the sample is usually fractionated first by IEF in a tube or strip gel to exploit the unique dependence of each protein's net charge on pH. Next, the gel containing the separated proteins is extruded from the tube, dried (these two steps can be bypassed using a strip gel) and laid horizontally along one edge of a slab gel, typically a crosslinked polyacrylamide gel containing sodium dodecylsulfate (SDS). Electrophoresis is then performed in the second dimension, perpendicular to the first, and the proteins separate on the basis of molecular weight. Thus, proteins having similar net charges, and which are not separated well in the first (IEF) dimension, will separate according to their different masses in the second dimension. Since these two separation methods depend on independent properties (net charge and mass), the overall resolution is approximately the product of the resolution in each dimension.
A significant drawback of traditional methods for two-dimensional electrophoresis is that two separate devices are used to accomplish electrophoresis in the two dimensions. These protocols can be very time-consuming and cumbersome to practice. Moreover, traditional methods are susceptible to significant run-to-run variation because of variability in standard IEF and SDS gels, which cannot be re-used.
Accordingly, there is a need for a new multidimensional electrophoresis method that is faster and easier to use, which allows the identification and characterization of hundreds or thousands of components in complex mixtures, and which is highly reproducible. Ideally, the method will employ a single separation apparatus for electrophoresis in both dimensions. The method preferably involves a flowable (liquid-state) separation medium that can be easily replaced with fresh media, so that a single apparatus can be used repetitively for multiple samples. Ideally, the apparatus is adaptable for automation.
SUMMARY OF THE INVENTION
The present invention is directed to methods and apparatus for conducting multidimensional electrophoresis of samples within a single apparatus, such that sample components that have been resolved in a first electrophoretic dimension can be directly electrophoresed in a second dimension that is substantially perpendicular to the first, without needing to move or manipulate the sample between the first and second electrophoretic steps.
In one aspect, the invention includes a two-dimensional electrophoresis system. In one embodiment, the system includes an electrophoresis plate assembly that defines (i) a sample separation cavity, (ii) a sample loading port positioned at a corner of the upper portion, for introducing a sample into the electrophoresis region, and (iii) optionally, one or more fluid passageways positioned along the lower portion of the cavity, for introducing or removing liquid from the cavity.
The cavity defined by the assembly is bounded by opposing major first and second surfaces, each having a defined width and length. These major surfaces are spaced apart by an interfacial distance substantially shorter than the width and length of the cavity. The cavity further comprises (1) a first electrophoresis region located along the upper portion of the cavity for performing charge and/or size-based electrophoresis in a first dimension along said upper portion, and (2) below the first electrophoresis region, a second electrophoresis region for performing electrophoresis in a second dimension in a direction substantially perpendicular to the first dimension, such that the rate of migration of sample components in the second dimension depends on sample properties that are different from the sample properties that determine the rate of migration in the first dimension.
In one embodiment, the second electrophoresis region is an isoelectric focusing region containing a continuous pKa gradient immobilized on at least one of the major opposing surfaces, for isoelectric focusing in a direction substantially perpendicular to the first dimension. The pKa gradient may span any attainable range, such as a pKa range of about 4 to 10, or 4 to 6, for example.
In another embodiment, the second electrophoresis region does not contain a pKa gradient and is used to perform charge and/or size-based electrophor
Raysberg Yefim
Wiktorowicz John E.
Gorthey LeeAnn
Ludlow Jan
Lynx Therapeutics, Inc.
Noguerola Alex
Powers Vincent M.
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