WO2004072236A2 - Methods and compositions for 3-d sodium dodecyl sulfate-polyacrylamide cube gel electrophoresis - Google Patents

Abstract

The present application is directed to methods and compositions for a three-dimensional sodium dodecyl sulfate polyacrylamide cube gel electrophoresis (3-D SDS-PACGE) technique to increase the resolution of protein separation. The three-dimensional technology involves the separation of proteins by isoelectric focusing in the first dimension, molecular weight separation using a high percentage SDS-PAGE in the second dimension, and molecular weight separation with a low percentage SDS-PACGE in the third dimension. In specific embodiments, one or more of the methods of the invention may employ a trapping agent that confers functionality to the separation dimension such that specific components of the mixture are bound to the trapping agent during the separation. Methods, apparati and compositions for performing 3-D SDS-PACGE are described.

Description

METHODS AND COMPOSITIONS FOR 3-D SODIUM DODECYL SULFATE-POLYACRYLAMIDE CUBE GEL ELECTROPHORESIS

Field of Related Cases and Government Funding The present application claims the benefit of priority of U.S.

Provisional Application Serial No. 60/445,499, which was filed February 6, 2003 and is incorporated herein by reference in its entirety.

Field of the Invention

The invention relates to the field of electrophoretic separations of macromolecules and in particular, to a technique for the electrophoretic separation used in the analysis of proteins.

Background of the Invention

Electrophoresis is one of the most widely used separation techniques in the biologically related sciences. This technique separates molecular species such as peptides, proteins, and oligonucleotides (analytes) by causing them to migrate at different rates in a separation medium under the influence of an electric field. The separation medium can be a buffer solution, or a low to moderate concentration of an appropriate gelling agent such as agarose or polyacrylamide. When gel separation medium is used, separation of analytes is partly based on their molecular sizes as the analytes are sieved by the gel matrix. Smaller molecules move relatively more quickly than larger ones through a gel of a given pore size which depends in part on the concentration of the polymer in the gel.

Initially, the most common electrophoretic separation methods were those in which the molecular species to be separated were loaded onto a matrix such as for example, a polyacrylamide gel. The gel is then subjected to an electric field that allowed the migration only in one direction. Such one-dimensional separation typically resolves about 100 distinct zones per gel. However one dimensional electrophoresis results in relatively poor resolution of a mixture of proteins which has a broad range of molecular weights. Subsequently, two-dimensional polyacrylamide gel electrophoresis (2-

D SDS- PAGE) was developed which allows an improved resolution of the protein ^* mixture. 2-D SDS- PAGE is a powerful technique widely used for the separation of hundreds to thousands of proteins in protein mixtures in a single analysis (Gδrg, et al, Electrophoresis, 21:1037-53, 2000). Conventional two-dimensional gel electrophoresis-based protein separation methods comprise two separation dimensions: isoelectric focusing, ("EEF") and sodium dodecyl sulfate-polyacrylamide gel electrophoresis ("SDS-PAGE").

IEF is almost exclusively the first separation dimension, h IEF, amphoteric molecules such as proteins are separated by electrophoresis in a pH gradient generated between a cathode and an anode. IEF takes advantage of the fact that each protein has a characteristic pH at which it is electrically neutral. This characteristic pH is the isoelectric point (pi) of the protein. Under the influence of an electric field, charged sample components migrate through an electrophoresis medium (a solution or a gel). If a sample component has a net negative charge, it migrates towards the anode. During migration, the negatively charged sample encounters a progressively lower pH, thus becoming more positively charged. Eventually, the pi is reached where the net charge of the sample component is zero. At the pi, migration stops and the sample component is "focused" in a tight zone. Likewise, if a sample component is positively charged, it will migrate towards the cathode. In this manner, each sample component migrates to its isoelectric point. LEF is a true electric field mediated focusing technique since protein molecules that diffuse out of the focused zone acquire charge and are pulled back into the zone where the net charge is zero.

The pH gradient, which is key to the success of IEF, is provided by molecules called "carrier ampholites". Carrier ampholites are polyamino- polycarboxic acids having gradually differing pi values. Ampholite mixtures are available in various nanow and broad pH ranges. Typically, an anti-convective media such as polyacrylamide or agarose is used. It is also possible to immobilize pH gradients on a suitable matrix such as polyacrylamide or ampholite strips. With immobilized pH gradients, LEF routinely provides a resolution of 0.1 to 0.01 pi units.

Relatively high electric field strengths are necessary to obtain rapid isoelectric focusing. Use of capillary dimensions (i.e. dimensions less than 0.2 mm ID.) provides efficient dissipation of Joule heat and permits the use of such high field strengths. In capillary dimensions, IEF separations can be carried out in free solution or in entangled polymer networks. After the focusing step, the second separation dimension in 2-D SDS PAGE is typically carried out by SDS-PAGE. SDS-PAGE involves complex relationships among several factors. These factors include separation length, gel composition, gel pore size, electric field strength, ionic moiety, buffer composition and the mode of migration of the polyion through the gel matrix. In conventional SDS-PAGE separations, biopolymers migrate under the influence of an electric field by tumbling through pores whose average radii are much larger that the radius of gyration of the analyte. Migrating samples are thereby size-ordered based on the time required to find a path through the pores of the gel matrix. This type of migration is known as separation in the Ogston regime, and is usually quite time-consuming. Larger molecules, i.e. those molecules whose radii of gyration are larger than the average pore size, are impeded and become oriented towards the electric field while migrating through the pores. This process can be induced through increases in either the gel concentration or the applied electric field strength. The use of increased electric field strengths (typically greater than lOOV/cm) necessitates thickness reduction in planar systems. Thickness reduction enhances the ability to dissipate heat and thereby reduces the effects of Joule heat. Some emerging capillary electrophoresis methods employ nanow-bore capillary columns having large surface-to-volume ratios to effectively dissipate heat, hi planar electrophoretic systems, the surface-to-volume ratio is increased through thickness reduction, ideally converging towards capillary dimensions. This is known as "ultra- thin" gel electrophoresis. Rapid biopolymer separation, for example, requires gel- filled separation platforms having a thickness of no more than 0.25 mm. The use of 0.1 mm thick gels for biopolymer separation allows as much as a five-fold increase in electric field strength. Use of polyacrylamide gels having a thickness of 0.025 to 0.1 mm permits resolution of complex mixtures of DNA sequencing reactions in less than 30 minutes.

The most recent advances in electrophoretic separation have been in methods such as capillary electrophoresis and in novel composite separation matrices. First, crosslinked polyacrylamide-polyethylene glycol copolymers were used to achieve size separation of SDS-protein molecules. Later, linear polymers such as non-crosslinked polyacrylamide, dextran and polyethylene oxides were shown to be effective, on a basis of chain-length, when subjected to an electric field. The use of non-crosslinked polymers has been primarily in high performance capillary electrophoresis applications, although high concentrations of non-crosslinked polymers can be used in planar formats to obtain separation of restriction fragments. Use of non-crosslinked polymers is advantageous in several respects. Non- crosslinked polymers may be supplied in a dessicated dry form, thereby providing a practically unlimited shelf life. Planar non-crosslinked polymer gels can be easily re- hydrated to any final gel concentration, buffer composition or strength.

Recently, the power of resolving as many proteins as possible in a single analysis has been shown to improve diagnostic accuracy of ovarian cancer (Petricoin III et al, Lancet, 359:572-7, 2002). Also, usefulness of the proteomics technique on yeast protein complexes [Gavin et al, Nature, 415:141-7, 4, 2002; Ho et al, Nature, 415:180-3, 2002) and identifications of cancer markers (Hondermarck et al, Proteomics, 1:1216-32, 2001) has been demonstrated. However, while 2-D SDS- PAGE is a versatile separation technique, there is still a great need for improvement in separation technology to provide greater resolution in protein separation in burgeoning field of proteomics.

The separation length necessary for resolution of protein molecules in planar ultra-thin gel electrophoresis is constantly being adjusted downward. To date, efforts to increase the resolution of protein separation have focused on improving gels for IEF and SDS-PAGE. Immobilized pH gradient (JJP G) gel with nanow pH range for the IEF has enhanced the resolution of protein separation by pi (Langen et al , Life Science News 4:6-8, 2000). Gradient gels and NuPAGE® gels for the SDS- PAGE gel with a variety of gel running buffers have provided further enhancement of the resolution of protein separation by Mw (Molecular Biology Catalog, pp. 649-669]. Nonetheless, these advances still leave room for improvement in the resolution of high molecular weight proteins in a given sample. Summary of the Invention

The present invention is directed to a three-dimensional SDS PAGE technique for the separation of proteins refened to herein as 3-D sodium dodecyl sulfate-polyacrylamide cube gel electrophoresis (SDS PACGE). The first dimension involves the separation of proteins by isoelectric point (pi), the second dimension involves the separation of the focused proteins from the first dimension by molecular weight with a high percentage SDS-PAGE, and the third dimension employs a second SDS-PAGE separation method by which the high molecular with proteins are separated by molecular weight with a low percentage SDS-PACGE. The utilization of two SDS-PAGE steps in 3-D SDS-PACGE compared to one single step in 2-D SDS-PAGE has increased the separation between proteins in a single analysis. The high percentage SDS-PAGE enhances the separation of the low molecular weight proteins. The low percentage SDS-PAGE enhances the separation of the high molecular weight proteins. In other embodiments, in one or more of the dimensions of separation the proteins are separated according to given functional characteristics.

In specific embodiments, the present disclosure is directed to a three- dimensional electrophoresis apparatus for the separation of the components of a mixture which comprises a three dimensional separation medium in which the components of the material mixture to be separated are at least partially spatially resolved according to one or more characteristics of the components of the mixture by migration under the influence of an electrical field along a first dimension of the separation medium; the partially spatially resolved by migration along the first dimension are further spatially resolved by migration under the influence of an electrical field along a second dimension of the separation medium under conditions different from those pertaining to migration along the first dimension; and the components of the material mixture to be separated that have been at least partially spatially resolved by migration along the first and second dimensions are yet further spatially resolved by migration under the influence of an electrical field along a third dimension of the separation medium under conditions different from those pertaining to migration along the first and second dimensions. The apparatus preferably contains a source for supplying an electrical field across opposed faces of the first, second or third dimension of the separation medium in accord with the dimension along which separation of the components of the mixture is presently being performed. The apparatus will be provided with a source that supplies electrical potentials and cunents suitable for establishing the desired electrical fields across opposed faces of the separation medium. The apparatus also may contain a mode for the in-situ detection of the spatially resolved material mixture wherein said the apparatus contains a detector positioned adjacent to the third dimension of the separation medium.

Generally, it is contemplated that the separation media of the first, and/or second, and/or third dimensions comprises an anticonvective separation medium selected from the group consisting of a gelatinous crosslinked polymer, a solution of one or more non-crosslinked linear polymers, a suspension of one or more non-crosslinked linear polymers and a porous membrane. Preferably, the anticonvective separation medium of the first, second and third dimensions comprises a crosslinked acrylamide — methylene-bis-acrylamide copolymer gel. In specific embodiments, the anticonvective separation medium of the first, second and third dimensions comprises an agarose gel.

The apparatus of the invention is used in a method in which the component mixture to be resolved is initially applied to one or more locations on the first dimension of the separation medium. Alternatively, the apparatus may be used in a method in which the component mixture to be resolved is incorporated into the medium of the first dimension.

The apparatus of the present invention preferably employs a pH gradient in the first dimension. Preferably the pH gradient is established between two opposed faces of the first dimension of separation, and the pH gradient resolves the components of the material mixture to the separated on the basis of the isoelectric points of the individual components. In specific embodiments, such a pH gradient is established by use of canier ampholytes. In other specific embodiments, the pH gradient is immobilized within the medium of the first dimension. Such prefened embodiments may be further characterized in that the first dimension is in the form of an immobilized pH gradient (IPG) gel. The three-dimensional electrophoresis apparatus of the invention is used in methods in which the spatial resolution of the components of the material mixture occurs in the second and third dimensions on the basis of the hydrodynamic radii of the components wherein the media of the second and third dimensions differ in the resistance offered to the migration of components of the mixture each component having a different hydrodynamic radius within some range of radii. Typically such separation is exemplified by PAGE, and more specifically SDS PAGE.

The three-dimensional electrophoresis apparatus of invention is preferably one which is used in a method in which the spatial resolution of the components of the material mixture occurs in the on the basis of the different strengths of binding interactions between individual components of the mixture and the solid components of the second dimension of the separation medium More specifically, the solid components of the second dimension of the separation medium comprise structurally incorporated therein chemical entities that preferentially bind to a specific component or group of components of the mixture over other components of the mixture. Specific entities that may be used to confer functionality to the dimension of separation include antibodies specific for a given component in the mixture to be separated.

In the three-dimensional electrophoresis apparatus described herein, the relative resistances to the migration of components of the mixture in the second and third directions is determined and controlled by the concentrations of the monomer(s) and crosslinking agent(s) employed in the preparation of the separation media in said second and third directions. More specifically in prefened ^• embodiments, the medium of the second dimension may comprise an acrylamide- methylene-bis-acrylamide gel containing between about 10% and 18% and preferably about 12% acrylamide. The medium of the third dimension preferably comprises an acrylamide-methylene-bis-acrylamide gel containing between about 3% and 10% and preferably about 7.5% acrylamide.

The apparatus of the present invention is particularly contemplated for use in methods for the separation of materials in which the component mixture to be resolved is comprised of complexes of protein with the detergent sodium dodecylsulfate (SDS) formed under conditions where the charge to mass ratios of the complexes are nominally the same or similar and where the hydrodynamic radii of the complexes are a nominal function of the molecular weight of the protein forming the complex. As indicated herein throughout, the apparatus of the present invention preferably comprises a detection device wherein the spatially resolved components are detected optically. Preferably, the detection device is such that it is able to detect optical contrast between resolved components and the separation medium. Such a contrast may be created or enhanced by means of one or more staining dyes selected from among chromatic dyes, chromogenic dyes, fluorescent dyes, and fluorogenic dyes. In such methods, the mixture to be resolved may preferably be treated with one or more staining dyes prior to being resolved. In certain embodiments, some or all of the components of the mixture to be resolved are exposed to, and bind or react with, a staining dye dispersed throughout the separation medium during the course of resolution. In specific methods, the mixture components resolved in a second dimension are preferably treated with a staining dye before being resolved in a third dimension.

Particularly prefened staining dyes include but are not limited to Procion and Ramazol dyes; textile industry Diazo chloromercury reactive dyes, and naphthoic disulfide dyes which react with cysteine. More specifically, the dyes include Uniblue A, Remazol Brilliant Violet, Reactive Blue 4, Reactive Blue 5, Reactive Blue 2, Reactive Orange 16, Reactive Orange 14, Reactive yellow 86, Reactive Green 5, Reactive Green 19, Reactive Brown 10, Reactive Red 120, Procion yellow H-E3G.

In specific embodiments, the detectors used in the apparatus of the invention include for example, fiber optics-based, laser-induced fluorescence systems. Alternatively, the detector is a Polaroid and/or a video monitor.

The apparatus of the invention preferably comprises a detector that comprises a source that emits light at a wavelength or within a wavelength band that is absorbed by the staining dye or dyes; a lens set for focusing light from the source onto the detection area; and a charge coupled device (CCD) based hyper-spectral image capture and analysis system.

In specific embodiments, the three-dimensional electrophoresis , apparatus is one in which the resolution of the components of a mixture in a first dimension is performed within a discrete linear separation medium that is subsequently placed into contact with a second discrete separation medium within which resolution in a second and a third dimension is performed. In other specific embodiments, the apparatus is one in which the resolution of the components of a mixture in a first and a second dimension is performed within a discrete planar separation medium that is subsequently placed into contact with a second discrete separation medium within which resolution on a third dimension is performed.

The present invention further contemplates a method for spatially resolving a mixture of proteins within an anisotropic separation medium, wherein the proteins are resolved by migration under the influence of an electrical field along a first dimension of the separation medium, said migration being modulated by the characteristics of the separation medium in that dimension; the proteins partially resolved by migration along the first dimension of the separation medium are then further resolved by migration under the influence of an electrical field along a second dimension of the separation medium, the migration along the second dimension being modulated by characteristics of the separation medium in the second dimension that differ from the characteristics in the first dimension; the proteins partially resolved by migration along a first and a second dimension of the separation medium are then further resolved by migration under the influence of an electrical field along a third dimension of the separation medium, the migration along the third dimension being modulated by characteristics of the separation medium in the third dimension that differ from the characteristics in the first and second dimensions; and detecting the spatial locations of the resulting protein zones within the three-dimensional volume of the separation medium. Preferably, the resolution along the first dimension of the separation medium is on the basis of the isoelectric points of the proteins comprising the mixture. Preferably, the method of the invention comprises performing a resolution along the second dimension of the separation medium which is performed on the basis of the hydrodynamic radii of the proteins comprising the mixture, the characteristics of the separation medium in said second dimension significantly restrict the migration of the proteins within the mixture that have large hydrodynamic radii relative to those of other proteins within the mixture. Alternatively, or additionally, the resolution along the third dimension of the separation medium is on the basis of the hydrodynamic radii of the proteins comprising the mixture, the characteristics of the separation medium in said third dimension being less restrictive to the migration of the proteins within the mixture that have large hydrodynamic radii than is the separation medium in the second dimension.

In specific embodiments, the method is one which is used to resolve a protein mixture that is comprised of complexes of the proteins with the detergent sodium dodecylsulfate (SDS) formed under conditions where the charge to mass ratios of the complexes are nominally the same or similar and where the hydrodynamic radii of the complexes are a nominal function of the molecular weights of the proteins forming complexes. Preferably, proteins within the mixture being resolved specifically bind to a moiety that is incorporated into the structure of the second dimension of the separation medium. In the methods of the invention, the detection of the resolved proteins preferably comprises staining the proteins with one or more staining dyes, illuminating the stained proteins with light of one or more wavelengths that are absorbed by the staining dyes and optically visualizing the stained proteins. Preferably, the staining dyes are chromatic dyes typically employed in the visualizing of proteins and detection comprises the measurement of the diminution of the illuminating light intensity resulting from the absorbance of the light by staining dye attached to the proteins. In other embodiments, the staining dyes are fluorescent and detection comprises the measurement of the fluorescent light emitted by staining dye(s) attached to the proteins. Preferably the staining dye is dispersed throughout one or more of the dimensions of the separation medium. More specifically, the dye is preferably dispersed through the second dimension of the separation medium and it binds to the proteins as they are resolved by migration through said second dimension of the separation medium. In particularly prefened embodiments, the proteins are treated with the staining dye before being subjected to resolution in the third dimensions.

While the methods described herein are exemplified for the separation of protein mixtures it should be understood that the methods may be used to effect the resolution of nucleic acid mixtures.

Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating prefened embodiments of the invention, are given by way of illustration only, because various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Brief Description of the Drawings The following drawings form part of the present specification and are included to further illustrate aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

Figure 1. Apparatus of three-dimensional sodium dodecyl sulfate polyacrylamide cube gel electrophoresis (3-D SDS-PACGE). (A) Cube gel cell with internal dimensions of 3 ^"(l) x 3 y₈"(w) x 3 W'Qa) with a thickness of %". (B) Cube gel bottom tray with internal dimensions of 3 ⁵/s"(l) x 3 ⁵/s"(w) x 1 ^"(h) with a thickness of V". (C) The assembly of the 3-D SDS-PACGE apparatus. (D) The copper screen cathode of the 3-D SDS-PACGE with dimensions of 3" x 3" with 15 x 15 lines per sq. in. and a wire diameter of 0.01 in. (E) The bottom cube gel cell holder pieces with dimensions of 3 "(1) x ³ t"(w) x A"( ). Cube was run at 10 watts constant.

Figure 2. (A) 12% NuPAGE® Bis-Tris 1-D SDS-PAGE with MES running buffer, (B) 7.5% Tris-Glycine 1-D SDS-PAGE, and (C) side view of 3-D SDS-PACGE of the wide range multi-colored protein Mw standard. The standard contains myosin (rabbit muscle, 205 kDa, iris blue), phosphorylase B ( rabbit muscle, 111 kDa, outrigger orange), glutamic dehydrogenase (bovine liver, 52 kDa, magenta), carbonic anhydrase (bovine erythrocytes, 34 kDa, hi-gloss Phoenician purple), myoglobin blue (horse heart, 19 kDa, flat Venetian blue), myoglobin red (horse heart, 17 kDa, pink), lysozyme (chicken egg white, 11 kDa, slicker yellow), aprotinin( bovine milk, 6,000 Da, pink), and insulin( bovine, 3,000 Da, hi-gloss true blue). Anows point to the higher Mw. Thirty μl of the multi-colored protein Mw standard with approximately total colored protein concentration of 5 mg/ml were loaded to each sample well. Figure 3. (A) Bird's-eye view of the 3-D SDS-PACGE and (B) side view of the 3-D SDS-PACGE of bovine serum albumin. First dimension, LPG 3-10; separation distance, 7 cm. Second dimension, vertical 12% NuPAGE® Bis-Tris SDS- PAGE; running buffer, MES. Third dimension, 7.5% Tris-Glycine SDS-PACGE; cube gel dimension, 3 ^"(l) x 3 ! g"(w) x 2 ^"(h). Arrows point to the higher Mw and higher pi values.

Figure 4 Separation of biotinylated insulin (20 μg) and biotinylated protein A (20 μg) by functionality using a 5% Tris-Glycine native polyacrylamide gel. An arrow indicates a 15 % trapping gel piece. Lane 1 shows a control gel which does not have a 15 % trapping gel piece; lane 2 has a 15 % trapping gel piece with Human IgG (100 μg) as the trapping agent; lane 3 has a 15 % trapping gel piece with Avidin (100 μg) as the trapping agent; lane 4 has a 15 % trapping gel piece without any protein trapping agent.

Detailed Description of the Preferred Embodiments of the Invention

2-D SDS PAGE has been known for many years as a versatile technique for the separation of proteins. However, where a sample being examined contains a large number of proteins with a wide range of molecular weights, 2-D SDS PAGE is ineffective at producing adequate and efficient resolution of the protein mixture.

Prior to the present invention, when additional resolution of the proteins in a complex mixture was required after the 2-D SDS PAGE step, individual gel bands from the SDS-PAGE gel of interest were excised, treated with chemicals, and then run on another separate 1-D gel (Dernick et al, Protides Biological Fluids, 977-982, 1985; Liang et al, Mol. Cell Biol. 14: 1520-1529, 1994). For example, the method of Liang et al. involves running a neutral/neutral 2D gel and then cutting out vertical gel slices from the area containing the protein sample of interest, rotating these excised slices 90 degrees to form the third dimension, and ranning an alkaline gel for each of the slices. However, these methods are labor-intensive and are extremely difficult to adapt to analysis of a large number of proteins as each of these methods require significant manipulation of the semi-separated proteins after the initial 2D separation. Moreover, such manipulation introduces the possibility of losses of quantity and contamination of the sample. The present invention describes methods and compositions for a three-dimensional sodium dodecyl sulfate polyacrylamide cube gel electrophoresis (3-D SDS-PACGE) technique which overcomes the prior cumbersome methods and provides better protein resolution than is seen in 2-D SDS PAGE.

The present invention for the first time provides details of an enhanced method for the separation of proteins in a single analysis using SDS-PAGE. The improved resolution seen in the present invention is achieved by introducing a third dimension in addition to 2-D SDS-PAGE. In this 3-D SDS-PACGE, the first dimension focuses the proteins of a mixture using IEF, much like the first step in 2-D SDS-PAGE. The focused proteins are then subjected to a separation in a second dimension in which the SDS-PAGE is performed to achieve resolution of low Mw proteins in the mixture. Preferable the SDS-PAGE in the second dimension is a high percentage (e.g., 12 %) SDS-PAGE with 2-[N-Morpholino]ethanesulfonic acid (MES) running buffer. The third dimension of the 3-D SDS-PACGE involves subjecting the gel containing the separated proteins from the second dimension to a second SDS-PAGE step in order to achieve resolution of high Mw proteins in the protein mixture. This third dimension is preferably carried out in a low percentage (e.g., 7.5 %) SDS-PAGE cube gel with Tris-Glycine gel running buffer.

The utilization of two SDS-PAGE steps in 3-D SDS-PACGE compared to one single step in 2-D SDS-PAGE has increased the separation between proteins in a single analysis. The high percentage SDS-PAGE enhances the separation of the low molecular weight proteins. The low percentage SDS-PAGE enhances the separation of the high molecular weight proteins. While prefened embodiments of the present application detail that the second dimension separation step is a high percentage SDS PAGE and the third dimension is a low percentage SDS PAGE separation, it should be understood that the order of these steps may be reversed. The examples and the description below describes the optimization of various steps including making and running of the cube gel, covalently coupling a colored dye to the proteins for visualization, and documenting the cube gel using a digital camera and a light box, and demonstrate the effectiveness of the 3-D SDS- PACGE technique.

Detailed Description of Preferred Apparatus and Method for Performing 3-D SDS-PACGE. The apparatus for performing 3-D SDS-PACGE is shown in Figure 1. The cube gel was made by a cell (Figure 1 A) of acrylic with a thickness of 0.25 inches and internal dimensions of 3.5 inches (length) x 3.5 inches (width) x 3.5 inches (height). Of course these are merely exemplary measurements for the cube and those of skill in the art would understand gels of other dimensions may be made and used according to the present invention. A copper screen (3 inches x 3 inches, 15 15 lines per square inch with a wire diameter of 0.01 in.) soldered to a copper wire was used as the cathode (Figure ID) of the 3-D SDS-PACGE. Figure IB shows the cube gel bottom tray with a thickness of 0.25 inches and internal dimensions of 3.625 inches (1) x 3.625 inches (width) x 1.25 inches (height) for holding the lower buffer. The anode of the 3-D SDS-PACGE is an 18 inch long platinum wire running the perimeter of the cube gel cell. There are two supporting pieces (Figure IE) of acrylic (3.25 inches (length) x 0.75 inches (width) x 0.125 inches (height)) to lift the electrophoresis cube gel cell so that the cube gel was not touching the bottom tray. PowerEase^® 500 programmable power supply (Invitrogen, Carlsbad, CA) was used as the power supply. The whole 3-D SDS-PACGE apparatus may be covered by a plastic box during the run for safety.

The cube gel of the 3-D SDS-PACGE was prepared as follows: A 7.5% polyacrylamide resolving gel solution (400 ml) was poured into the cube gel cell, which was sealed by a plastic plate on the bottom with universal box sealing tape, and allowed to polymerize. The 7.5% polyacrylamide resolving gel solution was prepared by mixing 100 ml 30.8% stock monomer acrylamide solution, 100 ml of 1.5 M Tris-HCl resolving gel buffer (pH 8.8), 4 ml of 10% SDS, 194 ml of ddH2O, 2 ml of APS (freshly prepared), and 133 μl of TEMED. Following gel formation, a 4% polyacrylamide stacking gel solution

(60 ml) was cast on top of the resolving gel and allowed to polymerize. The 4% polyacrylamide stacking gel solution was prepared by mixing 8 ml stock monomer acrylamide solution, 15 ml of 0.5 M Tris-HCl stacking gel buffer (pH 6.8), 0.6 ml of 10% SDS, 36 ml of ddH2O, 300 μl of APS (freshly prepared), and 30 ul of TEMED. Since air bubbles inside the cube gel are detrimental to the running of the 3-D SDS- PACGE, vacuum was applied to both resolving and stacking gel solutions while stirring on a magnetic stiner for several minutes to deaerate the solution. After the cube gel polymerized overnight, the sealing plastic plate was removed from the bottom and the cube gel cell was placed in the center of the bottom tray, and supported by the two supporting pieces. Fifty ml of gel running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) was added to the top of the cube gel cell (upper buffer reservoir) and 200 ml of gel running buffer was added to the bottom tray (lower buffer reservoir).

After completion of the 2-D gel run, the gel was laid on top of the cube gel (3.125 inches (length) x 3.125 inches (width) x 2.5 inches (h)). Electrophoresis was carried out at 10 watts with a cunent of ~250 mA and a voltage of approximately 40 volts for approximately 4 hours. The copper screen is used instead of copper plate as the cathode to prevent accumulation of air bubbles which will decrease the efficiency of the apparatus. Running the 3-D SDS-PACGE at higher wattage caused the cube gel to expand and crack due to a rise in gel temperature.

Staining of the cube gel with Coomassie blue or other dyes was difficult due to the difficulties of staining the proteins deep inside the cube gel and de- staining of the stained cube gel. Therefore, to visualize proteins, the proteins were covalently conjugated with colored dyes in the 2-D gel before the cube gel mn. Dyes that bind protein non-covalently including Coomassie blue were not as effective because the dyes dissociate from proteins during the cube gel ran.

The examples presented below show separation of bovine serum albumin and a broad range multi-colored protein Mw standard (Invitrogen). The use of a broad-range multi-colored protein Mw standard was chosen to show an easy visualization, a Mw calibration, and to demonstrate the efficacy and usefulness of the 3-D SDS-PACGE technique on wide range of molecular weights. Since multicolored protein Mw standard obtained from companies contained SDS and chemicals that are not suitable for IEF run, multi-colored protein standard was run directly on the 12% NuPAGE® Bis-Tris gel and then the cube gel without the IEF step. The bovine serum albumin was used as a control sample which was separated using all three steps. After the IEF and 12% SDS-PAGE steps, BSA (100 μg) in the 2-D SDS- PAGE gel were stained covalently with 20 ml freshly prepared Remazol Brilliant Violet 5R (10 mg/ml) at room temperature for 30 min. at pH 11 in 10 mM CAPS buffer and then de-stained in water for 15 min. This staining process was repeated three times before running the cube gel. The 2-D SDS-PAGE gels were stained covalently with 20 ml freshly prepared Uniblue A (5mg/ml) in water at room temperature for 30 min. before staining with 20 ml freshly prepared Remazol Brilliant Violet 5R (10 mg/ml) at room temperature for 30 min at pH 11 in 10 mM CAPS buffer. At the end of the 3-D SDS-PACGE run, cubes were pushed out and documented using a digital camera and a light box. Those of skill in the art are aware of the gel compositions and voltages commonly used in 2D SDS PAGE techniques. It should be understood that any of these compositions and experimental designs that are presently in use for 2D SDS PAGE may be adapted for 3D SDS PACGE. To the extent that additional description of materials and methods for performing 2D SDS PAGE is necessary, those of skill in the art are refened to, for example, Molecular Cloning: A Laboratory Manual, 3rd Edition, Vol.1-3, Sambrook et al., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, 2001. In addition, those of skill in the art are refened U.S. Patent No. 6,507,664 describes two-dimensional gels for use in electrophoresis methods; U.S Patent No. 6,398,933 describes a two dimensional electrophoresis system; U.S. Patent Nos 6,482,303; 6,480,618; 6,451,189 describes an automated system for use in two- dimensional electrophoresis; microchip devices for use in the operation and control of electrophoresis systems are described in U.S. Patent No. 6,319,705; protein sample preparation for electrophoresis is described in U.S. Patent No. 6,391,650; additional methods and sample preparations for 2D electrophoresis are described in U.S. Patent Nos. 6,245,206 6,277259, 6,214191; 5,562,813. Each of these U.S. Patents are incorporated herein by reference in their entirety. Methods and compositions that are used in the SDS-PAGE step of these conventional 2D SDS PAGE techniques maybe employed in either the second or the third dimensions of the 3D SDS PACGE methods and compositions described herein. In addition, described herein below are additional details that may be used to adapt the 3D SDS PACGE methods and compositions described herein.

B. Detailed Description of An Alternative Embodiment for the Three- Dimensional Separation of Proteins.

In addition to the 3D SDS PACGE methods of the present invention, it is contemplated that the second dimension may be modified such that proteins are separated according to given functional characteristics. In this alternative embodiment, the dimension of the "functionality" will preferably be the second dimension, however, it is contemplated that the third or even the first dimension may be the dimension on which the mixture is resolved according to functionality. This functionality dimension will be used to separate the complex protein mixture according to different functionalities such as glycoprotein, lipoprotein, phosphorylated protein, antibodies, and the like. Such separation may be achieved by preparing the matrix of the second dimension (e.g., low percentage native PAGE gel) with many layers of affinity tags that are specific for the given class of proteins to be separated. The affinity tags may be trapped in the native PAGE gel or covalently linked the gel matrix. Exemplary affinity tags for use in the second dimension include lectin for the separation of glycoproteins, antibodies against phosphorylated proteins, lipoproteins and the like.

In an exemplary embodiment, this 3D separation may comprise in the first dimension a conventional IEF separation matrix in order to separate proteins by PI. The second dimension is one designed specifically to receive and further separate proteins, which were separated in the first dimension, according to the functionality of the proteins in the complex mixture and the third dimension comprises an SDS PAGE matrix to effectuate the separation of proteins separated in the second, dimension according to molecular weight. The matrix for the second dimension may be polyacrylamide gel, agarose, a mixture of polyacrylamide and agarose or any other matrix to facilitate the separation of proteins. Preferably the matrix for the second dimension is a polyacrylamide gel native gel without SDS. Agents that will bind to the proteins of interest, e.g., substrates or affinity tags for the proteins are trapped in a high percentage area of the gel using conventional polyacrylamide gel preparation techniques well known to those of skill in the art. The remainder of the gel is a low percentage gel so protein can pass freely. While the proteins are passing through this area, only proteins that bind with the substrates with be retained as they bind to the affinity tags. In order to trap the affinity tags in the polyacrylamide gel, the affinity tags are placed into a solution before the acrylamide is polymerized. Substrates can be trapped covalently by copolymerization with the acrylamide. In order to covalently trap the affinity tag, the tag is labeled with acrylic acid n-hydroxy- succinimide ester and copolymerized with acrylamide.

An exemplary embodiment of this alternative 3D separation system in which one of the three dimensions is based on separation according to the functional groups or other affinity type separation of the proteins, this "functional separation" dimension is set up as follows. In the referenced exemplary embodiment, about 10 ml of the resolving gel solution was poured into a 10 x 10 cm empty cassette with a thickness of 1 mm (Invitrogen). A comb was inserted on the top of the cassette to wells. The 5% polyacrylamide resolving gel solution was prepared by mixing 2.5 ml 30.8% stock monomer acrylamide solution, 3.75 ml of 1.5 M Tris-HCl resolving gel buffer (pH 8.8), 8.7 ml of ddH2O, 75 μl of APS (freshly prepared), and 5 μl of TEMED. Following gel formation and comb removed, 30 μl of 15% trapping gel solution which contains no protein, Avidin, or Human IgG was loaded on top of each well. Trapping gel solution was prepared by mixing 15 μl stock monomer acrylamide solution, 7.5 μl of 1.5 M Tris-HCl resolving gel buffer (pH 8.8), 7.4 μl of ddH2O, 1 μl of APS (freshly prepared), and 0.1 μl of TEMED. In this example, the trapping moieties used to retain the proteins are avidin and human IgG, however, it should be understood that those of skill will be able to modify the separation technique by using a trapping moiety other than avidin, e.g., antibodies specific for phosphorylation status, glycoprotein-trapping moieties, ligands for receptors etc.

The results of the separation of proteins in this dimension are depicted in Figure 4. Lane 3 in Figure 4 which shows that when biotinylated insulin and biotinylated protein A are passed through a trapping gel that contains avidin, both biotinylated proteins are retained in the trapping gel. On the other hand, as can be seen in lane 2, when biotinylated insulin and biotinylated protein A are passed through a trapping gel that contains human IgG, only biotinylated protein A is retained in the trapping gel. Lane 4 shows that biotinylated insulin and biotinylated protein A are separated through the trapping gel, which contains no protein, no biotinylated proteins have been retained in the trapping gel. C. Isoelectric Focusing (IEF)

A protein is a macromolecule composed of a chain of amino acids. Of the 20 amino acids found in typical proteins, four (aspartic and glutamic acids, cysteine and tyrosine) cany a negative charge and three (lysine, arginine and histidine) a positive charge, in some pH range. A specific protein, defined by its specific sequence of amino acids, is thus likely to incorporate a number of charged groups along its length. The magnitude of the charge contributed by each amino acid is governed by the prevailing pH of the sunounding solution, and can vary from a minimum of 0 to a maximum of 1 charge (positive or negative depending on the amino acid), according to a titration curve relating charge and pH according to the pK of the amino acid in question. Under denaturing conditions in which all of the amino acids are exposed, the total charge of the protein molecule is given approximately by the sum of the charges of its component amino acids, all at the prevailing solution pH.

Two proteins having different ratios of charged, or titrating, amino acids can be separated by virtue of their different net charges at some pH. Under the influence of an applied electric field, a more highly charged protein will move faster than a less highly charged protein of similar size and shape. If the proteins are made to move from a sample zone through a non-convecting medium (typically a gel such as polyacrylamide), an electrophoretic separation will result. If, in the course of migrating under an applied electric field, a protein enters a region whose pH has that value at which the protein's net charge is zero (the isoelectric pH), it will cease to migrate relative to the medium. Further, if the migration occurs through a monotonic pH gradient, the protein will "focus" at this isoelectric pH value. If it moves toward more acidic pH values, the protein will become more positively charged, and a properly-oriented electric field will propel the protein back towards the isoelectric point. Likewise, if the protein moves towards more basic pH values, it will become more negatively charged, and the same field will push it back toward the isoelectric point. This separation process, called isoelectric focusing, can resolve two proteins differing by less than a single charged amino acid among hundreds in the respective sequences.

A key requirement for an isoelectric focusing procedure is the formation of an appropriate spatial pH gradient. This can be achieved either dynamically, by including a heterogeneous mixture of charged molecules (ampholytes) into an initially homogeneous separation medium, or statically, by incorporating a spatial gradient of titrating groups into the gel matrix through which the migration will occur. The former represents classical ampholyte-based isoelectric focusing, and the latter the more recently developed immobilized pH gradient (LPG) isoelectric focusing technique. The LPG approach has the advantage that the pH gradient is fixed in the gel, while the ampholyte-based approach is susceptible to positional drift as the ampholyte molecules move in the applied electric field. The best cunent methodology combines the two approaches to provide a system where the pH gradient is spatially fixed but small amounts of ampholytes are present to decrease the adsorption of proteins onto the charged gel matrix of the LPG

It is cunent practice to create IPG gels in a thin planar configuration bonded to an inert substrate, typically a sheet of Mylar plastic which has been treated so as to chemically bond to an acrylamide gel (e.g., Gelbond® PAG film, FMC Corporation). The LPG gel is typically formed as a rectangular plate 0.5 mm thick, 10 to 30 cm long (in the direction of separation) and about 10 cm wide. Multiple samples can be applied to such a gel in parallel lanes, with the attendant problem of diffusion of proteins between lanes producing cross contamination. In the case where it is important that all applied protein in a given lane is recovered in that lane (as is typically the case in 2-D electrophoresis), it has proven useful to split the gel into nanow strips (typically 3 mm wide), each of which can then be run as a separate gel. Since the protein of a sample is then confined to the volume of the gel represented by the single strip, it will all be recovered in that strip. Such strips (hnmobiline DryStrips) are produced commercially by Pharmacia Biotech.

While the nanow strip format solves the problem of containing samples within a recoverable, non-cross-contaminating region, there remain substantial problems associated with the introduction of sample proteins into the gel. Since protein-containing samples are typically prepared in a liquid fonn, the proteins they contain must migrate, under the influence of the electric field, from a liquid- holding region into the IPG gel in order to undergo separation. This is typically achieved by lightly pressing an open-bottomed rectangular frame against the planar gel surface so that the gel forms the bottom of an open-topped but otherwise liquid- tight vessel (the sample well). The sample is then deposited in this well in contact with the gel surface forming the bottom of the well. Since all of the sample protein must pass through a small area on the surface of the gel (the well bottom) in order to reach the gel interior, the local concentration of protein at the entry point can become very high, leading to protein precipitation. The sample entry area is typically smaller than the gel surface forming the well bottom because the protein migrates into the gel under the influence of an electric field which directs most of it to one edge of the well bottom, tending to produce protein precipitation. The major source of precipitation, however, is provided by the charged groups introduced into the gel matrix to form the pH gradient in IPG gels: these groups can interact with charges on the proteins (most of which are not at their isoelectric points at the position of the application point and hence have non-zero net charges) to bind precipitates to the gel. It is common experience that separations of the same protein mixture on a series of apparently identical LPG gels can yield very different quantitative recoveries of different proteins at their respective isoelectric points, indicating that the precipitation phenomenon may vary from gel to gel in unpredictable ways, thereby frustrating the general use of LPG gels for quantitative protein separations.

Recently, methods have been introduced in which the LPG strip is re- swollen, from the dry state, in a solution containing sample proteins, with the intention that the sample proteins completely permeate the gel at the start of the run. Isoelectric focusing separation of proteins in an immobilized pH gradient (LPG) is extensively described in the art. The concept of the LPG is disclosed in U.S. Pat. No. 4,130,470 and is further described in numerous publications. The LPG gel strips manufactured are generally of simple planar shape.

In addition to the above described gel configurations, there are various configurations of cavities ("sample wells") used for the application of macromolecular-containing samples to the surfaces of gels, most frequently slab gels used for protein or nucleic acid separations. These sample wells are generally designed to concentrate macromolecules in the sample into a thin starting zone prior to their migration through the resolving gel. The use of devices placed against a gel to form wells has been described for example in U.S. Patent No. 5,304,292, which describes the use of pieces of compressible gasket to form well walls at the top of a slab where the ends of the pieces touch the top edge of the slab. Additionally, U.S. Patent No. 5,164,065 describes a shark's tooth comb used in combination with DNA sequencing gels. Several references describe automated devices for creating gradients of polymerizable monomers. Such systems have been used for making porosity gradient gels used in molecular weight separations of proteins. Altland et al. (Clin. Chem. 30(12 Pt 1):2098-2103, 1984) shows the use of such systems for creating the gradients of titratable monomers used in the creation of LPG gels. U.S. Patent No. 4,169,036 describes a system for loading slab-gel holders for electrophoresis separation. U.S. Patent No. 4,594,064 discloses an automated apparatus for producing gradient gels. Hence, use of a computer-controlled gradient maker in manufacturing IPG and other gels is known in the art. Systems for making non-planar slab gels are also known in the art, e.g.,

U.S. Patent No. 5,074,981 discloses a substitute for submarine gels using an agarose block that is thicker at the ends and hangs into buffer reservoirs. U.S. Patent No. 5,275,710 discloses lane-shaped gels formed in a plate and gel- filled holes extending down from the plate into buffer reservoirs. Any of the methods for preparing IPG gels described in these patents listed in the present section may be adapted for the SDS PACGE methods of the present invention.

D. SDS Slab Gel Electrophoresis

The second and third dimensions of the SDS PACGE methods described herein employ PAGE based methods. In PAGE, charged detergents such as sodium dodecyl sulfate (SDS) can bind strongly to protein molecules and "unfold" them into semi-rigid rods whose lengths are proportional to the length of the polypeptide chain, and hence approximately proportional to molecular weight. While the methods of the present invention are directed to SDS-PACGE in which the inert matrix is polyacrylamide. It should be understood that those of skill in the art may adapt the methods to the use of other polymers including but not limited to non-cross linked polyacrylamide, dextran, polyethylene oxides, derivatized celluloses, polyvinylpynolidone and mixtures thereof.

A protein complexed with such a detergent is itself highly charged (because of the charges of the bound detergent molecules), and this charge causes the protein-detergent complex to move in an applied electric field. Furthermore, the total charge also is approximately proportional to molecular weight (since the detergent's charge vastly exceeds the protein's own intrinsic charge), and hence the charge per unit length of a protein-SDS complex is essentially independent of molecular weight. This feature gives protein-SDS complexes essentially equal electrophoretic mobility in a non-restrictive medium. If the migration occurs in a sieving medium, such as a polyacrylamide gel, however, large (long) molecules will be retarded compared to small (short) molecules, and a separation based approximately on molecular weight will be achieved. This is the principle of SDS electrophoresis as applied commonly to the analytical separation of proteins.

An important application of SDS electrophoresis involves the use of a slab-shaped electrophoresis gel as the second dimension of a two-dimensional procedure. The gel strip or cylinder in which the protein sample has been resolved by isoelectric focusing is placed along the slab gel edge and the molecules it contains are separated in the slab, perpendicular to the prior separation, to yield a two-dimensional (2-D) separation. Fortunately, the two parameters on which this 2-D separation is based, namely isoelectric point and mass, are almost completely unrelated. This means that the theoretical resolution of the 2-D system is the product of the resolutions of each of the constituent methods, which is in the range of 150 molecular species for both IEF and SDS electrophoresis. This gives a theoretical resolution for the complete system of 22,500 proteins, which accounts for the intense interest in this method. In practice, as many as 5,000 proteins have been resolved experimentally.

Those of skill in the art are refened to U.S. Patent No. 5,993,627, incorporated herein by reference in its entirety, provides teachings of an integrated, fully automated, high- throughput system for two-dimensional electrophoresis comprised of gel-making machines, gel processing machines, gel compositions and geometries, gel handling systems, sample preparation systems, software and methods. That patent provides a description of methods for automating the second dimension SDS separation of a 2-D process to afford higher throughput, resolution and speed. Such automation techniques are contemplated to be useful in the 3D separation processes of the present invention. It is cunent practice to mold electrophoresis slab gels between two flat glass plates, and then to load the sample and run the slab gel still between the same glass plates. The gel is molded by introducing a dissolved mixture of polymerizable monomers, catalyst and initiator into the cavity defined by the plates and spacers or gaskets sealing three sides. Polymerization of the monomers then produces the desired gel media. This process is typically carried out in a laboratory setting, in which a single individual prepares, loads and rans the gel. A gasket or form comprising the bottom of the molding cavity is removed after gel polymerization in order to allow cunent to pass through two opposite edges of the gel slab: one of these edges represents the open (top) surface of the gel cavity, and the other is formed against its removable bottom. Typically, the gel is removed from the cassette defined by the glass plates after the electrophoresis separation has taken place, for the purposes of staining, autoradiography, etc., required for detection of resolved macromolecules such as proteins.

The concentrations of polyacrylamide gels used in electrophoresis are stated generally in terms of %T (the total percentage of acrylamide in the gel by weight) and %C the proportion of the total acrylamide that is accounted for by the crosslinker used). N,N'-methylenebisacrylamide ("bis") is typically used as crosslinker. Typical gels used to resolve proteins range from 8% T to 24% T, a single gel often incorporating a gradient in order to resolve abroad range of protein molecular masses.

In the present invention it is contemplated that the second dimension separation preferably employs a high percentage SDS PAGE gel such as for example 12 % SDS-PAGE with 2-[N-Morpholino]ethanesulfonic acid (MES) running buffer to separate low Mw proteins. Those of skill in the art should be able to vary the percentage of the SDS-PAGE remain within the scope of the invention. Hence, any SDS-PAGE gel having greater than SDS PAGE and preferably between about 10% to about 24% SDS PAGE may be used for the second dimension. Similarly, running buffers other than MES that are typically used in running high percentage SDS PAGE systems may be used and include but are not limited to Tris glycine, Tris Page and the like.

The third dimension separation in the methods of the present invention is designed to separate the high molecular weight proteins in a sample and employs a lower percentage SDS PAGE composition than in employed in the second dimension. In prefened embodiments, the third dimension employs a 7.5% SDS-PAGE cube gel with Tris-Glycine gel running buffer. Again the percentage of the SDS PAGE cube gel may be varied between 0.1% to about 10% percent SDS PAGE cube gel configuration. The running buffer may be any buffer typically employed in running conventional low percentage SDS PAGE systems. Sambrook et al., supra, describes conditions for low and high molecular weight SDS PAGE systems at §§A8.40 through A8.51. For example, percentage ranges and the linear range of separation of proteins on such gels is given in table A8.8 therein and indicates that a 15% acrylamide gel will likely yield a linear separation of proteins in the range of 10 kDa to about 43 kDa; a 12% acrylamide gel will likely yield a linear separation of proteins in the range of 12 kDa to about 60 kDa; a 10% acrylamide gel will likely yield a linear separation of proteins in the range of 20 kDa to about 80 kDa; a 7.5% acrylamide gel will likely yield a linear separation of proteins in the range of 36kDa to about 94 kDa; and a 5% acrylamide gel will likely yield a linear separation of proteins in the range of 57 kDa to about 212 kDa. Thus, those of skill in the art. will be able to adapt the percentages of acrylamide in the SDS PACGE gels in accordance with particular needs using the teachings provided herein and those teachings readily available in the art. Specifically contemplated are gels in which the high percentage gel is either 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25% SDS PAGE and the low percentage gel is either 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% SDS PAGE. Any combination of these low and high percentage gels may be employed in the gel cube used in the present invention. In most conventional systems used for. SDS electrophoresis, use is made of the stacking phenomenon first employed in this context by Laemmli, U. K. (1970) Nature 227:680. In a stacking system, an additional gel phase of high porosity is interposed between the separating gel and the sample. The two gels initially contain a different mobile ion from the ion source (typically a liquid buffer reservoir) above them: the gels contain chloride (a high mobility ion) and the buffer reservoir contains glycine (a lower mobility ion, whose mobility is pH dependent). All phases contain Tris as the low-mobility, pH determining other buffer component and positive counter-ion. Negatively charged protein-SDS complexes present in the sample are electrophoresed first through the stacking gel at its pH of approximately 6.8, where the complexes have the same mobility as the boundary between the leading (C1-) and trailing (glycine-) ions. The proteins are thus stacked into a very thin zone "sandwiched" between CI- and glycine-zones. As this stacking boundary reaches the top of the separating gel the proteins become unstac ed because, at the higher separating gel pH (8.6), the protein-SDS complexes have a lower mobility. Thus, in the separating gel, the proteins fall behind the stacking front and are separated from one another according to size as they migrate through the sieving environment of the lower porosity (higher %T acrylamide) separating gel. In this environment, proteins are resolved on the basis of mass.

Pre-made slab gels have been available commercially for many years (e.g., from Integrated Separation Systems). These gels are prepared in glass cassettes much as would be made in the user's laboratory, and shipped from a factory to the user. More recently, methods have been devised for manufacture of both slab gels in plastic cassettes (thereby decreasing the weight and fragility of the cassettes) and slab gels bonded to a plastic backing (e.g., bonded to a Gelbond® Mylar® sheet or to a suitably derivatized glass plate). To date, all commercially-prepared gels are either enclosed in a cassette or bonded to a plastic sheet on one surface (the other being covered by a removable plastic membrane). Furthermore, all commercially-prepared gels have a planar geometry. The methods and compositions employed to prepare these pre-made gels also may be used in the preparation of the 3D cubes for the present invention.

Cunent practice in running slab gels involves one of two methods. A gel in a cassette is typically mounted on a suitable electrophoresis apparatus, so that one edge of the gel contacts a first buffer reservoir containing an electrode (typically a platinum wire) and the opposite gel edge contacts a second reservoir with a second electrode, steps being taken so that the cunent passing between the electrodes is confined to run mainly or exclusively through the gel. Such apparatus may be "vertical" in that the gel's upper edge is in contact with an upper buffer reservoir and the lower edge is in contact with a lower reservoir, or the gel may be rotated 90° about an axis perpendicular to its plane, so that the gel runs horizontally between a left and right buffer reservoir, as is disclosed in U.S. Pat. No. 4,088,561 (e.g., "DALT" electrophoresis tank). Various configurations have been devised in order to make these connections electrically, and to simultaneously prevent liquid leakage from one reservoir to the other (around the gel).

When used as part of a typical 2-D procedure, an IEF gel is applied along one exposed edge of such a slab gel and the proteins it contains migrate into the gel under the influence of an applied electric field. The IEF gel may be equilibrated with solutions containing SDS, buffer and thiol reducing agents prior to placement on the SDS gel, in order to ensure that the proteins the LEF gel contains are prepared to begin migrating under optimal conditions, or else this equilibration may be performed in situ by sunounding the gel with a solution or gel containing these components after it has been placed in position along the slab's edge.

A slab gel affixed to a Gelbond® sheet is typically run in a horizontal position, lying flat on a horizontal cooling plate with the Gelbond® sheet down and the unbonded surface up. Electrode wicks communicating with liquid buffer reservoirs, or bars of buffer-containing gel, are placed on opposite edges of the slab to make electrical connections for the nm, and samples are generally applied onto the top surface of the slab (as in the instractions for the Pharmacia ExcelGels).

The proteins in 2-D gels generally are detected either by staining the gels or by exposing the gels to a radiosensitive film or plate (in the case of radioactively labeled proteins). Staining methods include dye-binding (e.g. Coomassie Brilliant Blue), silver stains (in which silver grains are formed in protein- containing zones), negative stains in which, for example, SDS is precipitated by Zn ions in regions where protein is absent, or the proteins may be fluorescently labeled. In each case, images of separated protein spot patterns can be acquired by scanners, and this data reduced to provide positional and quantitative information on sample protein composition through the action of suitable computer software. In the context of the present invention, the dyes used to stain the proteins should preferably be those that for strong, e.g., covalent bonds with the proteins in the 3D cube. Such dyes include but are not limited to, reactive dyes such as Procion and Ramazol dyes; textile industry Diazo chloromercury-type reactive dyes, dyes react with phenolic or naphthoic protein moiety, and naphthoic disulfide dyes which react with cystine may be used. Those of skill in the art are refened to Floyd J. Green "The Sigma- Aldrich Handbook of Stains, Dyes and indicators" 1990 ISBN 0-941633-22-5, Aldrich Chemical Company, Inc., Milwaukee, Wisconsin for additional dyes that may be used in the present invention. In addition, Griffin et al. (Science 281, 269-272, 1998) describe the specific covalent bonding of proteins using 4',5'-bis(l,3,2-dithioarsolan- 2-yl) Fluorescein, an agent which is nonfluorescent until it binds a protein containing tetracysteine domains. In addition to conventional covalent fluorophore labeling, the use of fluorescent affinity ligands in the separation medium expands detection sensitivity and separation potential. The positive charge of complexing dye molecules (e.g., Ethidium Bromide, Sypro dyes) can significantly affect the migration velocity of the protein molecules relative to the biopolymer-stain complex. Hence, the complex formation with fluorescent stain permits high sensitivity fluorescence- detection of the migrating protein molecules and can be utilized to achieve higher resolution.

The methods disclosed herein can be used for a series of alternative analytical applications including the analysis of DNA and RNA, as well as peptides. Automation procedures for the 3D SDS PACGE akin to the automations of the 2D SDS PAGE presently being employed are contemplated.

E. Examples

The following examples are included to demonstrate prefened embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention,, and thus can be considered to constitute prefened modes for its practice. However, those, of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 Separation of BSA and a Broad Range of Molecular Weight Proteins

The bovine serum albumin and a broad range multi-colored protein Mw standard (Invitrogen) were used to provide evidence of the efficacy and usefulness of the 3-D SDS PACGE technique. This Example demonstrates that a mixture of proteins of a broad range of molecular weights can be separated using the instant technique. Since multi-colored protein Mw standard obtained from companies contained SDS and chemicals that are not suitable for LEF ran, multi-colored protein standard was run directly on the 12% NuPAGE® Bis-Tris gel and then the cube gel without the IEF step. The bovine serum albumin was however used as a control sample that was subjected to all three steps. After the LEF and 12% SDS-PAGE steps, BSA (100 μg) in the 2-D SDS-PAGE gel were stained covalently with 20 ml freshly prepared Remazol Brilliant Violet 5R (10 mg/ml) at room temperature for 30 min. at pH 11 in 10 mM CAPS buffer and then destained in water for 15 min. This staining process was repeated three times before running the cube gel. At the end of the 3-D SDS-PACGE ran, cubes were pushed out and documented using a digital camera and a light box.

The separation of a broad range multi-colored protein Mw standard using the 3-D SDS-PACGE and 1-D SDS-PAGE are shown in Figure 2. The side views of the cube gel ran (Figures 2C) shows the 12 % SDS-PAGE (MES running buffer) dimension and the 7.5 % SDS-PAGE (Tris-Glycine ranning buffer) dimension. The IEF dimension was not used. The conesponding 12% 1-D SDS- PAGE and 7.5% 1-D SDS-PAGE are shown in Figure 2A and Figure 2B, respectively. A direct comparison between the cube gel and the 1-D SDS-PAGE gels can be made. All proteins are resolved with the conect molecular weights by the cube gel. The cube gel exhibits resolution of the protein separation of both the 12 % SDS- PAGE and 7.5 % SDS-PAGE gels in a single experiment run.

The cube gel ran of the broad range protein standard showed that the separation between phosphorylase B and glutamic dehydrogenase is enhanced in the 7.5 % SDS-PAGE dimension because the pore size of the 7.5% SDS-PAGE gel is optimized to separate high Mw proteins. Further, the separations of the low Mw proteins myoglobin blue, myoglobin red, lysozyme, aprotinin, and insulin are enhanced with the 12 % SDS-PAGE dimension because the pore size of the 12% SDS-PAGE gel is optimized to separate low Mw proteins. Interestingly, the two bands of myoglobin are reversed in the NuPAGE^® Bis-Tris system compared to the Tris-Glycine system, the separation between myosin and phosphorylase B is about the same between these two type of SDS-PAGE gels, and there is very little separation between glutamic dehydrogenase and carbonic anhydrase in the 7.5 % SDS-PAGE dimension. It is known that differences in protein mobilities occur when the same proteins are ran in different SDS-PAGE buffer systems (Molecular Biology Catalog, pp. 649-669, 10, 2002), Patton et al, Anal Biochemistry 197:25-33, 1991). This changes the apparent Mw and account for the observation seen here.

Figure 3 shows the 3-D SDS-PACGE results of BSA. Figure 3A shows a bird's-eye view of the cube gel with pi 3-10 as the first dimension and 12% SDS-PAGE as the second dimension. Figure 3B shows a side view of the cube gel with 12% SDS-PAGE as the first dimension and 7.5% SDS-PAGE as the second dimension. The 3-D SDS-PACGE exhibits resolution of the protein separation of both the 12 % SDS-PAGE and the 7.5 % SDS-PAGE gels in a single experiment run. Figure 3 clearly shows the enhancement of the separation of the BSA and the high Mw contamination bands in a single experiment ran.

Example 2 Separation of Proteins According to Functionality

As discussed herein above in Section B, the second dimension of separation may employs the "functionality" to separate the components of a given mixture according to different functionalities such as glycoprotein, lipoprotein, phosphorylated protein, antibodies, and the like. Such separation may be achieved by preparing the matrix of the second dimension (e.g., low percentage native PAGE gel) with many layers of affinity tags that are specific for the given class of proteins to be separated. The affinity tags may be trapped in the native PAGE gel or covalently linked the gel matrix. The present Example provide one exemplary separation of proteins using avidin, anti-BSA antibodies and concanavalin A incorporated into a 7.5% native PAGE. Using such antibodies embedded in the PAGE matrix, it is possible to anest the antigens at the site where the antigen-specific antibody is embedded.

Materials & Methods

All chemicals were purchased from Sigma (Saint Louis, MO), Invitrogen (Carlsbad, CA), Pierce (Rockford, IL), and Research Organics Inc. (Cleveland, OH) and used without further purification except monoclonal antibody. Monoclonal antibody was purified by a HiTrap rProtein A affinity column (5 X 1 ml, Amersham Bioscience) chromatography. Capacity human IgG: 50 mg/ml gel specificity is Fc region. niAb from ascites and cell culture supernatants. The binding buffer was 20 mM sodium phosphate, pH7. The elution buffer was 0.1 M sodium citrate, pH 3-6. For purification of low binding IgG up to 4 M NaCl is added to the binding buffer. The high binding buffer was 20 mM sodium phosphate, 3 M NaCl, pH 7. High binding buffer: 1.5 M glycine, 3 M NaCl, pH 8.9. The unpurified antibody is filtered through a 0.45 μm filter or centrifuged immediately before it is applied to the column. The flow rate was 1 ml/min and the collection tubes contained 60-100 μl of 1 M Tris-HCl, pH 9 per ml of fraction to be collected. The syringe or pump tubing is filled with starting buffer and the column is equilibrated with 5-10 column volumes of binding buffer.

The sample to be separated is applied to the column using a syringe fitted to the luer adaptor or by pumping the sample onto the column. The column is then washed with 5 column volumes of binding buffer. The antibody is eluted with 2- 5 column volumes of elution buffer. The column is then washed five times with 20% ethanol to prevent microbial growth. The purified IgG fractions can be desalted by buffer exchange using a PD-10 desalting column. The 1-D polyacrylamide electrophoresis were prepared and run according to the manufacturer's (Invitrogen, Pharmacia) protocols.

The functional affinity electrophoresis (FAEP) gel was prepared as follows: A 7.5% polyacrylamide resolving gel solution (8 ml) was poured into a empty gel cassette (Invitrogen) with plastic strips positioned at a depth of 1/3 from the top of the gel to form the wells. The plastic strips have dimensions of 100 mm (1) x 8 mm (w) x 10 mm (h). The 7.5% polyacrylamide resolving gel solution was prepared by mixing 7.5 ml 30.8% stock monomer acrylamide solution (Sigma), 7.5 ml of 1.5 M Tris-HCl 4 x resolving gel buffer (pH 8.8), 12.9 ml of ddH2O, 150 μl of 10% APS (ammonium persulfate solution), and 10 μl of TEMED (tetramethylethylenediamine). Following gel formation and removal of the plastic strips, 50 μl of the 7.5% polyacrylamide gel solution with or without 200 μg of avidin was added to the wells and allowed to polymerize. 75 μl of 7.5% polyacrylamide gel was poured and allow to polymerized. Another 75 μl of 280 μg of anti-BSA, another 75 μl of 500 μg con A to complete the wells. Since air bubbles in the gel are detrimental to the running of the FAEP, vacuum was applied to resolving gel solutions while stirring on a magnetic stiner for several minutes to deaerate the solution. 500 ml of gel running buffer (25 mM Tris, 192 mM glycine, pH 8.3) was used for the gel ran. PowerEase® 500 programmable power supply (Invitrogen, Carlsbad, CA) was used as the power supply. Electrophoresis was canied out at 3 watts with a current of ~22 mA and a voltage of ~125 volts for ~ 1.5 hours. All the gels were stained with coomassie blue G-250 and documented using a digital camera and a light box.

The FAEP was performed to separate biotinylated insulin, BSA, and ovalbumin. These proteins were chosen because they carried negative charges while avidin carries a positive charge at the pH of the gel running buffer - 8.3. The ID SDS-PAGE was used to detect the components of the avidin-biotinylated protein complex. The complex was excised from the 5% CCAE gel and soaked in the SDS sample loading buffer to dissociate the avidimbiotinylated protein complex and the gel piece was loaded on the 4-12% SDS-NuPage gel (Invitrogen) with 2-[N- Morpholinojethanesulfonic acid (MES) running buffer. Pierce' s TriChroRanger molecular weight marker was used to provide a MW calibration.

To further confirm the components of the complex, the complex was excised and subjected to trypsin in-gel digestion. For the digestion, the protein gel was washed with 50% acetonitrile in 50 mM ammonium bicarbonate pH 8.0 and was shrunk with neat acetonitrile. The shrunk gel was then dried in speedvac. Reduction of the disulfide bond and alkylation of the free sulfhydryl group were done simultaneously by DTT (dithiothreitol) and 4-vinylpyridine in 6 M guanidine HCI 50 mM ammonium bicarbonate pH 8.0 with 5 mM EDTA , respectively. After alkylation, gel was washed with 50 mM ammonium bicarbonate pH 8.0 and then shrank with acetronitrile. The shrank gel was then dried in speedvac. Trypsin (Sigma) in 50 mM ammonium bicarbonate pH 8.0 solution at a concentration of 2ug/100 μl was then added to the dried gel. Digestions were performed at 37 °C for 24 h. The digestion solution was extracted. The residual gel peptides were further extracted with a 50% water/50% acetonitrile solution containing a final concentration of 5% trifluoroacetic acid (TFA). All peptide extracts were pooled.

ZipTips, packed with C18 resin, were used to prepare the in-gel digestion for MS analysis using cyano-4-hydroxycinnamic Acid (CHCA) as the matrix. Samples were spotted onto a 182-position MALDI-TOF (matrix-assisted laser desorption/ionization time-of- flight) target and analyzed by a positive-ion Voyager DE-PRO Mass Spectrometer (Applied Biosystems) equipped with a nitrogen laser. Peptide mass maps were measured at an instrument resolution of 10,000 in a reflector mode with delayed extraction over the m/z range 700-4000 Da. Samples were externally calibrated. Computer softwares from protein prospector, available from the University of California at San Francisco (http://prospector.ucsf.edu), were used to interpret the MS results. Results and Discussion

Affinity electrophoresis has been used successfully for years in glycoprotein studies (Shariff and Parija, J. Microbiol. Methods 14 (1991) 71-761; Kurata and Tan, Arthritits Rheum 19 (1976) 574-580). In these studies the sugar is covalently bind to the polyacrylamide and either pack in the gel or make a gel which can retard the movement of the analyte and allow the calculation of the binding constant. However, since the ligand is permanently fixed with the gel, the interaction force is not strong enough to counteract the electrophoresis force of migration. Usually, what had been observed is a retardation of the migration and from this retardation kinetic parameters can be calculated. It is hard to observe the effect during the electrophoresis migration and it is dependent on the strength of the interaction.

Affinity electrophoresis have been used successfully to retard or even stop glycoproteins, plasma proteins, enzymes, nucleic acids, ϊectins, receptors, and extracellular matrix proteins by the specific interactions with their ligands during electrophoretic migration in support media with a little molecular sieving effect (Shariff and Parija, J. Microbiol Methods 14 (1991) 71-761; Kurata and Tan, Arthritits Rheum 19 (1976) 574-580). The FAEP method described above was used to separate proteins by their functions based on the principles of affinity electrophoresis and electroimmunodiffusion. The avidin :biotin, con A:ovalbumin, and anti-BSA antibody:BSA complexes were used as a model system.

The avidin, anti-BSA, and con A are incorporated into separate layers of a 7.5% native PAGE gel. Avidin (66 kDa) with a basic isoelectric point (pi) of 10.5 migrates upward at the pH of the tris-glycine gel running buffer. Anti-BSA (PI 8.4, MW 150 kDa) and con A (PI 5.1, MW 80 kDa) move downward slowly. A protein mixture of Biotinylated insulin (bovine pancreas, PI 5.3, 6 kDa, 1 mole biotin per mole insulin), biotinylated bovine serum albumin (BSA, 66 kDa, pi 5.5, 8-16 moles biotin per mole BSA), ovalbumin (chicken, 45 kDa, pi 5.1), and myoglobin (horse heart, 18 kDa, pi 7) were used in this study. The results showed that biotinylated proteins stop and precipitate at the equivalence zone and non-biotinylated proteins migrate freely. Ovalbumin stopped at the con A zone due to the selective binding of con A with mannopyranosyl residues. BSA stopped at the anti-BSA ' antibody zone. Mass spectrometry and sodium dodecyl sulfate polyacrymide (SDS- PAGE) were used to further confirm the formation of avidin :biotin, bsa:antiBSA (bonding constant), ovalbuminxon A (bonding constant) complexes. Protein A and protein G with human anti-a lactobumin.

In the mass spectrometry analysis, peptide fragments at 2698 Da and 2924 Da were observed and were annotated as being from biotinylated insulin. Peaks at 1228 Da, 1440 Da, 1567 Da, 1663 Da, and 1774 Da were from biotinylated BSA, and a peak at 819 Da is from avidin after trypsin digestion of the protein complex gel. These results indicate that the complex was composed of avidin, biotinylated insulin, and biotinylated BSA. Interestingly, the peak at 2924 Da is the biotinylated peptide of 2698 Da of amino acid residue 1 to 21 of the biotinylated insulin chain B (a mass difference of 226). Peaks at 1228 Da, 1470 Da, 1663 Da, and many others are the biotinylated peptides of biotinylated BSA.

Example 3 Further Example of Separation of Proteins According to Functionality

The methods described in Example 2 were modified with a different set of functionalities to effect separation of a mixture of proteins. In this Example, Anti-ubiquitin, anti-BSA, and anti-GST antibodies were incorporated into a 7.5% native PAGE. Under gel running conditions, these antibodies do not move much compare to the analytes. The results of this separation showed that ubiquitin stops migrating in the gel when it encounters anti-ubiquitin, BSA stops migrating when it encounters anti-BSA antibody, and GST stops its migrating upon encountering anti- GST.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of prefened embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

The references cited herein throughout, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are all specifically incorporated herein by reference.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A three-dimensional electrophoresis apparatus for the separation of the components of a mixture comprising a three dimensional separation medium, wherein said medium comprises: a) a first dimension of said separation medium for the spatially resolution of the components of said mixture according to one or more characteristics of the components of the mixture by migration under the influence of an electrical field along a first dimension of the separation medium; b) a second dimension of said separation medium that receives and separates the components separated in step (a), wherein said second dimension comprises a separation medium to further separate said separated components of step (a) according to one or more characteristics of the components of the mixture by migration under the influence of an electrical field along said second dimension of the separation medium; c) a third dimension of said separation medium that receives and separates the components separated in step (b), wherein said third dimension comprises a medium to further separate said separated components of step (b) according to one or more characteristics of the components of the mixture by migration under the influence of an electrical field along said third dimension of the separation medium, wherein the separation conditions employed in said third dimension are different from those pertaining to migration along the first and second dimensions; d) a power supply for applying an electrical field across opposed faces of the first, second or third dimension of the separation medium in accord with the dimension along which separation of the components of the mixture is presently being performed; and e) a detection system for the in-situ detection of the spatially resolved material mixture, wherein said detection means is positioned adjacent to the third dimension of the separation medium

2. The three-dimensional method of claim 1 , wherein the separation media of the first, second and third dimensions comprises an anticonvective separation medium selected from among: a gelatinous crosslinked polymer, a solution of one or more non-crosslinked linear polymers, a suspension of one or more non-crosslinked linear polymers and a porous membrane.

3. The three-dimensional electrophoresis apparatus of claim 2, wherein the anticonvective separation medium of the first, second and third dimensions comprises a crosslinked acrylamide - methylene-bis-acrylamide copolymer gel.

4. The three-dimensional electrophoresis apparatus of claim 2, wherein the anticonvective separation medium of the first, second and third dimensions comprises an agarose gel.

5. The. three-dimensional electrophoresis apparatus of claim 1, wherein the component mixture to be resolved is incorporated into the medium comprising the first dimension.

6. The three-dimensional electrophoresis apparatus of claim 1, wherein said first dimension of separation comprises a pH gradient for resolving the components of the material mixture to be separated on the basis of the isoelectric points of the individual components of said mixture.

7. The three-dimensional electrophoresis apparatus of claim 6, wherein. the pH gradient is established by carrier ampholytes.

8. The three-dimensional electrophoresis apparatus of claim 6, wherein pH gradient is immobilized within the medium of said first dimension.

9. The three-dimensional electrophoresis apparatus of claim 8, wherein the first dimension is in the form of an immobilized pH gradient (IPG) gel.

10. The three-dimensional electrophoresis apparatus of claim 1, wherein said second and said third dimension comprise media for resolving proteins in said mixture according to the relative resistance to migration of said proteins in said media.

11. The three-dimensional electrophoresis apparatus of claim 1 , wherein said second and said third dimension comprise SDS-PAGE media for resolving proteins.

12. The three-dimensional electrophoresis apparatus of claim 1, wherein the media of said second or said third dimension comprises a moiety that specifically interacts with and binds an individual component of the mixture.

13. The three-dimensional electrophoresis apparatus of claim 10, wherein the relative resistances to the migration of components of the mixture in the second and third directions is determined and controlled by the concentrations of the monomer(s) and crosslinking agent(s) employed in the preparation of the separation media in said second and third directions.

14. The three-dimensional electrophoresis apparatus of claim 13, wherein the medium of the second dimension comprises an acrylamide-methylene- bis-acrylamide gel that comprises between about 10% and about 18% acrylamide.

15. The three-dimensional electrophoresis apparatus of claim 14, wherein the medium of the second dimension comprises an acrylamide-methylene- bis-acrylamide gel that comprises about 12% acrylamide.

16. The three-dimensional electrophoresis apparatus of claim 14, wherein the medium of the third dimension comprises an acrylamide-methylene-bis- acrylamide gel that comprises between about 3% and about 10% acrylamide.

17. The three-dimensional electrophoresis apparatus of claim 16, wherein the medium of the third dimension comprises an acrylamide-methylene-bis- acrylamide gel that comprises about 7.5% acrylamide.

18. The three-dimensional electrophoresis apparatus of claim 1, wherein the component mixture is prepared in a medium comprising the detergent sodium dodecylsulfate.

19. The three-dimensional electrophoresis apparatus of claim 1, wherein the spatially resolved components are detected spatially.

20. The three-dimensional electrophoresis apparatus of claim 1, wherein said detection system comprises a fiber optics-based laser induced fluorescence system.

21. The three-dimensional electrophoresis apparatus of claim 1, wherein said detection system comprises a Polaroid camera and videotaping apparatus.

22. The three-dimensional electrophoresis apparatus of claim 1, wherein said detection system comprises a) a source that emits light at a wavelength or within a wavelength band that is absorbed by the staining dye or dyes; b) a lens set for focusing light from the source onto a detection area; c) a charge coupled device (CCD) based hyper-spectral image capture and analysis system.

23. The three-dimensional electrophoresis apparatus of claim 1, wherein the resolution of the components of a mixture in a first dimension is perfonned within a discrete linear separation medium that is subsequently placed into contact with a second discrete separation medium within which resolution in a second and a third dimension is performed.

24. The three-dimensional electrophoresis apparatus of claim 1, wherein the resolution of the components of a mixture in a first and a second dimension is performed within a discrete planar separation medium that is subsequently placed into contact with a second discrete separation medium within which resolution on a third dimension is performed.

25. A method for spatially resolving a mixture of proteins within an anisotropic separation medium, comprising: a) resolving the proteins by migrating the proteins under the influence of an electrical field along the first dimension of the separation medium of claim 1, said migration being modulated by the characteristics of the separation medium in that dimension to producing a partially resolved mixture of proteins; b) further resolving the partially resolved mixture of proteins of step (a) by migrating the proteins under the influence of an electrical field along the second dimension of the separation medium of claim 1, said migration being modulated by characteristics of the separation medium in the second dimension that differ from the characteristics in the first dimension; o c) further resolving the protein mixture resolved in step (b) by migrating the mixture under the influence of an electrical field along the third dimension of the separation medium of claim 1, said migration being modulated by characteristics of the separation medium in the third dimension that differ from the characteristics in the first and second dimensions; and d) detecting the spatial locations of the resulting protein zones within the three-dimensional volume of the separation medium.

26. The method of claim 25, wherein resolution along the first dimension of the separation medium is on the basis of the isoelectric points of the proteins of said mixture.

27. The method of claim 25, wherein resolution along said second dimension comprises resolving said proteins on an acrylamide-methylene-bis- acrylamide gel that comprises between about 10% and about 18% acrylamide.

28. The method of claim 27, wherein resolution along said second dimension comprises resolving said proteins on an acrylamide-methylene-bis- acrylamide gel comprises about 12% acrylamide.

29. The method of claim 25, wherein resolution along said third dimension comprises resolving said proteins on an acrylamide-methylene-bis- acrylamide gel comprises between about 3% and about 10% acrylamide.

30. The method 29, wherein resolution along said third dimension comprises resolving said proteins on an acrylamide-methylene-bis-acrylamide gel comprises about 7.5% acrylamide.

31. The method of claim 27 or 28, wherein said gel comprises a moiety that preferentially binds to a component of said protein mixture.

32. The method of claim 29 or 30, wherein said gel comprises a moiety that preferentially binds to a component of said protein mixture.

33. The method of claim 25, wherein said method comprises creating or enhancing the optical contrast between resolved components and the separation medium by contacting the protein mixture with one or more staining dyes selected from the group consisting of a chromatic dye, a chromogenic dye, a fluorescent dye, and a fluorogenic dye.

34. The method of claim 33, wherein said mixture to be resolved is contacted with one or more staining dyes prior to being resolved in said method.

35. The method of claim 33, wherein said dye is dispersed throughout at least one dimension of said separation medium.

36. The method of claim 33, wherein the protein mixture is contacted with said dye after said components have been resolved in said second dimension and prior to resolution in said third dimension.

37. The method of claim 33, wherein said staining dye is selected from the group consisting of Procion and Ramazol dyes; textile industry Diazo - chloromercury reactive dyes, and naphthoic disulfide dyes which react with cysteine.

38. The method of claim 33, wherein said dye is selected from the group consisting of Uniblue A, Remazol Brilliant Violet, Reactive Blue 4, Reactive Blue 5, Reactive Blue 2, Reactive Orange 16, Reactive Orange 14, Reactive yellow 86, Reactive Green 5, Reactive Green 19, Reactive Brown 10, Reactive Red 120, and Procion yellow H-E3G.

39. The method of claim 25, wherein said detection comprises: a) treating the proteins with one or more staining dyes; b) illuminating the stained proteins with light of one or more wavelengths that are absorbed by the staining dyes; and c) optically visualizing the resolved, stained proteins in a spatially resolved manner.

40. The method of claim 39, wherein the staining dyes are chromatic and said detection comprises the measurement of the diminution of the illuminating light intensity resulting from the absorbance of the light by staining dye attached to the proteins.

41. The method of claim 39, wherein the staining dyes are fluorescent and said detection comprises the measurement of the fluorescent light emitted by staining dye(s) attached to the proteins.

42 The method of claim 39, wherein the staining dye is dispersed throughout the second dimension of the separation medium and binds to the proteins as they are resolved by migration through said second dimension of the separation medium

43. The method of claim 39, wherein the proteins are treated with the staining dye before being subjected to resolution in the third dimension.

44. A method of producing a differential profile of the protein composition of a diseased cell as compared to a normal cell comprising: a) performing the method of any of claims 25 through 43 on a mixture of proteins of a normal cell; b) performing the method of any of claims 25 through 43 on a mixture of proteins of a diseased cell of the same cell type as said normal cell; and c) comparing the spatial distributions produced by the resolution of the proteins of normal and diseased cells within the separation medium to identify differences in the expression and/or processing of proteins in normal and diseased cells.

45. A three-dimensional SDS polyacrylamide cube gel electrophoresis (3-D SDS-PACGE) apparatus comprising: a separation cassette for providing three-dimensional separation of a protein sample, wherein the cassette includes a first dimension separation compartment housing a material having capillary channels, the protein sample being disposed in the capillary channels for first dimension separation; a second dimension separation compartment housing a separation medium, the separation medium receiving the protein sample from the first dimension for second dimension separation; a third dimension separation compartment housing a separation medium, the separation medium receiving the protein sample from the second dimension for third dimension separation; a power supply configured to apply an electric field across either the first dimension compartment, the second dimension compartment, or the third dimension; an illumination and detection system positioned adjacent the third dimension separation compartment for illuminating and detecting the separated protein sample during third dimension separation; and an analysis system for processing data received from the illumination and detection system and formatting the data into a three-dimensional map representing the separated protein sample.

46. The 3-D SDS-PACGE apparatus of claim 45, further comprising a material for creating a pH gradient disposed in the second dimension separation compartment.

47. The 3-D SDS-PACGE apparatus of claim 45, wherein the material having capillary channels is a porous membrane.

48. The 3-D SDS-PACGE apparatus of claim 45 , wherein the material of the first dimension is an LPG strip.

49. The 3-D SDS-PACGE apparatus of claim 45, wherein the material having capillary channels is an inert matrix.

50. The 3-D SDS-PACGE apparatus of claim 45, wherein the second dimension separation compartment comprises a high percentage SDS PAGE gel greater than 10% SDS.

51. The 3-D SDS-PACGE apparatus of claim 50, wherein the second dimension separation compartment comprises a high percentage SDS PAGE gel of between about 10% to about 18% SDS PAGE gel

52. The 3-D SDS-PACGE apparatus of claim 50, the SDS PAGE gel is a 12% SDS-PAGE gel.

53. The 3-D SDS-PACGE apparatus of claim 45, wherein the third dimension separation compartment comprises a low percentage SDS PAGE gel of between about 3% to about 10% SDS.

54. The 3-D SDS-PACGE apparatus of claim 45, wherein the third dimension separation compartment comprises a 7.5% SDS-PAGE gel.

55. The 3-D SDS-PACGE apparatus of claim 7, further comprising a staining dye disposed in the separation medium.

56. The 3-D SDS-PACGE apparatus of claim 45, wherein the illumination and detection system is an integrated fiber optics-based, laser-induced fluorescence detection system.

57. The 3-D SDS PACGE apparatus of claim 56, wherein the illumination and detection system for the detection of a light emitting label comprises:

a laser for emitting an illuminating beam; a lens set for focusing the illuminating beam on the detection area; an excitation fiber for transmitting the illuminating beam from the laser to the lens set; a translation stage for oscillating the lens set over the detection area; collection fibers for collecting fluorescent light emitted by the separated protein sample; and an avalanche photodiode detector for receiving the collected light from a label.

58. The 3-D SDS-PACGE apparatus of claim 45, wherein the illumination and detection system comprises a CCD camera-based hyper-spectral imaging system.

59. The 3-D SDS-PACGE apparatus of claim 45, wherein the analysis system is a PC having data analysis software.

60. A separation cassette for providing three-dimensional separation comprising:

first, second and third reservoirs, wherein the first reservoir is a first dimension separation compartment and contains a porous material having capillary channels, a protein sample disposed in the porous material, and a pH gradient;

a second dimension separation compartment fluidly connected to the first and second reservoirs, the second dimension compartment comprising two glass or plastic plates separated by an ultra-thin layer of a linear polymer suspended in an inert matrix;

a third dimension separation compartment fluidly connected to the first and second reservoirs, the third dimension compartment comprising two glass or plastic plates separated by an ultra-thin layer of a linear polymer suspended in an inert matrix; and

a power supply configured to apply an electric field across either the first reservoir to effect isolectric focusing or across the second dimension separation compartment to effect separation by a sieving effect.

61. The separation cassette of claim 60, further comprising an illumination and detection system and an analysis system, the illumination and detection system comprising:

a light source for emitting an illuminating beam; a lens set for focusing the illuminating beam on a detection area of the third dimension separation compartment; an excitation fiber for transmitting the illuminating beam from the laser to the lens set; a translation stage for oscillating the lens set over the detection area; collection fibers for collecting fluorescent light emitted by the separated protein sample; and an avalanche photodiode detector for receiving the collected fluorescent light and supplying data to the analysis system.

62. A method for analyzing a mixed protein sample by three- dimensional separation comprising:

(a) disposing the protein sample in a material having capillary channels;

(b) disposing a material having a pH gradient in the capillary channels;

(c) applying a first electric field to the material to effect a first dimension separation by isoelectric focusing of components of the protein sample;

(d) contacting the material containing the focused protein sample with a first separation medium;

(e) applying a second electric field to the separation medium to effect a second dimension separation of the protein sample;

(f) contacting the material containing the separated protein sample from step (e) with a second separation medium;

(g) applying a third electric field to the separation medium to effect a third dimension separation of the protein sample through the second separation medium;

(h) labeling the protein sample, wherein a staining dye incorporated into the separation medium forms a complex with the protein sample; (i) detecting the presence of the labeled protein material in the protein sample; and

(j) analyzing the data from the detection performed in step (g) to produce a 3 -dimensional separation map of the separated protein sample.

63. The method of claim 62, wherein said labeling of the protein sample comprises fluorescent labeling and said detection comprises illumination and collection of the light emitted from the fluorescently labeled sample.

64. The method of claim 62, wherein in step (a), the protein sample is absorbed into a porous membrane having capillary channels.

65. The method of claim 62, wherein in step (b), the pH gradient is absorbed into the porous membrane.

66. The method of claim 62, wherein in step (a), the protein sample is injected into an inert matrix having capillary channels.

67. The method of claim 62, further comprising the step of labeling the protein sample before said third dimension separation.

68. A method for analyzing a protein sample by three-dimensional separation comprising:

(a) disposing the protein sample in a material having capillary channels;

(b) disposing a material having a pH gradient in the capillary channels;

(c) applying a first electric field to the material to effect a first dimension separation by isoelectric focusing of components of the protein sample; (d) contacting the material containing the focused protein sample with a first SDS PAGE separation medium;

(e) applying a second electric field to the separation medium to effect a second dimension separation of the protein sample;

(f) contacting the material containing the separated protein sample from the first SDS PAGE separation of step (e) with a second SDS PAGE separation medium;

(g) applying a third electric field to the separation medium to effect a third dimension separation of the protein sample;

(h) covalently labeling the protein sample;

(i) detecting the presence of the covalently labeled proteins;

(j) analyzing the detected labels and formatting a three-dimensional image map conesponding to the separated protein sample; wherein steps (i) and (j) are preformed simultaneously with step (g).

69. A method for analyzing a protein sample by three dimensional separation comprising:

(a) performing two dimensional SDS-PAGE on said protein sample in a three-dimensional cube gel, wherein the first dimension of the two dimensional SDS PAGE comprises IEF of the protein sample and the second dimension of the two- dimensional SDS PAGE comprises a high percentage SDS PAGE separation of the proteins focused by the IEF to produce a focused and separated protein sample; and

(b) subjecting the focused and separated protein sample to a third dimension separation, wherein the third dimension separation comprises a low percentage SDS PAGE separation.

70. The method of claim 69, further comprising covalently labeling the protein sample before one or more of the first, second or third dimension separation steps.

71. The method of claim 70, wherein the protein sample is labeled with only one label.

72. The method of claim 70, wherein the protein sample is labeled with a plurality of labels.

73. A method for analyzing a protein sample by three dimensional separation comprising:

(a) subjecting the protein sample to IEF focusing to effect a first dimension separation of the protein sample;

(b) subjecting the material containing the focused protein sample to a first SDS-PAGE separation to effect a second dimension separation of the protein sample;

(c) subjecting the material containing the focused and separated protein sample of step (b) to a second SDS-PAGE separation to effect a third dimension separation of the protein sample;

(d) covalently labeling the protein sample; and

(e) collecting and analyzing the data from the covalent labeling pattern of the proteins to produce a three-dimensional image map conesponding to the separated protein sample; wherein steps step (c) is performed without excising protein samples from step (b).

74. A method for spatially resolving a mixture of nucleic acids within an anisotropic separation medium, comprising: a) resolving the mixture by migrating the nucleic acids under the influence of an electrical field along the first dimension of the separation medium of claim 1, said migration being modulated by the characteristics of the separation medium in that dimension to producing a partially resolved mixture of nucleic acids; b) further resolving the partially resolved mixture of nucleic acids of step (a) by migrating the nucleic acids under the influence of an electrical field along the second dimension of the separation medium of claim 1, said migration being modulated by characteristics of the separation medium in the second dimension that differ from the characteristics in the first dimension; c) further resolving the nucleic acid mixture resolved in step (b) by migrating the mixture under the influence of an electrical field along the third dimension of the separation medium of claim 1, said migration being modulated by characteristics of the separation medium in the third dimension that differ from the characteristics in the first and second dimensions; and d) detecting the spatial locations of the resulting nucleic acid zones within the three-dimensional volume of the separation medium.