Abstract:
A device for two-dimensional electrophoresis includes a cassette comprising two opposing plates. The two opposing plates form a first elongated portion for receiving a first elongated electrophoretic separation medium and a second portion extending away from the first portion. A second electrophoretic separation medium is on the second portion and between the two opposing plates. A dialysis membrane extends across the second electrophoretic separation medium.

Description:
BACKGROUND OF THE INVENTION 
   The present invention relates to the field of performing two-dimensional electrophoresis. 
   Electrophoresis using 2-D PAGE (Two-dimensional PolyAcrylamide Gel Electrophoresis) techniques can separate proteins in a sample according to isoelectric pH (IpH) and molecular weight. Two-Dimensional PAGE generally provides isoelectric focusing electrophoresis in one direction followed by polyacrylamide gel electrophoresis in a second direction, and can be useful in analyzing the protein composition of a sample solution such as a biological sample. For example, gene activity may be studied by analyzing various proteins important to certain cellular functions. 
   For example, isoelectric focusing can use Immobilized pH gradient (IPG) strips to separate proteins based on their respective IpH values. An IpH is the pH value at which a protein carries no net charge and will not migrate in an electric field. An IPG strip with zwitter ionic peptides fixed to its surface can establish an identifiable pH gradient when a voltage is applied to electrodes on opposite sides of the IPG strip. Therefore, when a protein sample is applied to an IPG strip, each protein in the sample travels through the IPG strip until it reaches its IpH value on the IPG strip. 
   After proteins from the sample are focused on the IPG strip, a buffer, such as a buffer including SDS (sodium dodecyl sulfate), can be applied in preparation for polyacrylamide gel electrophoresis. Sodium dodecyl sulfate is a detergent that can solubize proteins to generate a uniform negative charge. Therefore, the SDS buffer disrupts hydrophobic interactions, increases the solubility of the protein, and leaves the protein molecules negatively charged. As a result, when the proteins are exposed to a polyacrylamide gel to which an electric current is applied, the proteins travel through the polyacrylamide gel and are separated according to their molecular weight. The polyacrylamide gel is typically a flat planar gel slab supported by a cassette housing. 
   After completion of the isoelectric focusing in one direction and PAGE in a second direction, the polyacrylamide gel has concentrations of protein deposits that are separated by IpH in one direction and molecular weight in the other direction. In order to view the resulting protein deposits, the polyacrylamide gel can be stained, for example, by removing the gel from its cassette housing and applying a staining reagent such as a silver or ruby protein stain. 
   Despite producing highly resolved results, 2-D PAGE presents technical challenges that may result in low reproducibility of results. The number of manual steps involved in 2-D PAGE may require a high level of operator skill to produce reliable 2-D PAGE results. In addition to being a labor-intensive process, many technical difficulties can be caused by operator inconsistencies. For example, the isoelectric focusing steps and the polyacrylamide electrophoresis steps are often carried out using separate devices, which may introduce poor reproducibility due to operator inconsistencies. U.S. Patent Application Publication No. 2002/0100690 to Herbert (hereinafter “Herbert”), disclosure of which is hereby incorporated by reference in its entirety, proposes a method in which the IPG strip and an agarose gel slab are carried on a single cassette. Increased automation of the 2-D PAGE steps may improve reproducibility of results and decrease the need for highly skilled operators. 
   SUMMARY OF THE INVENTION 
   Methods, systems, and devices for electrophoresis are provided that may address some of the challenges discussed above. Embodiments of the present invention provide a device for two-dimensional electrophoresis including a cassette comprising two opposing plates. The two opposing plates form a first elongated portion for receiving a first elongated electrophoretic separation medium and a second portion extending away from the first portion. A second electrophoretic separation medium is on the second portion and between the two opposing plates. A dialysis membrane extends across the second electrophoretic separation medium. 
   In some embodiments, the second electrophoretic separation medium can be deposited on one of the plates, and the dialysis membrane can define a void between the dialysis membrane and the other of the two opposing plates. The void can be used to inject fluids onto the electrophoretic separation media without requiring that the electrophoretic separation media be removed from the cassette. 
   In other embodiments, a loading chamber for an electrophoresis device is provided comprising a sample chamber having and opening configured to release a sample solution from the sample chamber into an cassette. A hydration chamber is adjacent the sample chamber, and a semi-permeable membrane is between the sample chamber and the hydration chamber. The membrane allows osmotic diffusion of fluid between the sample chamber and the hydration chamber through the membrane. Loading chambers according to embodiments of the present invention can be used to concentrate a protein sample solution prior to electrophoresis. 
   In further embodiments, methods for staining an electrophoresis cassette include providing an electrophoresis cassette comprising first and second opposing plates. An electrophoresis medium and a membrane layer on the electrophoresis medium are provided between the first and second opposing plates such that a void is formed between the membrane layer and the first opposing plate. Electrophoresis separation of a sample solution is conducted using the electrophoresis medium. A staining reagent is applied to the electrophoresis medium through the void between the membrane layer and the first opposing plate. A staining solution may be automatically injected into the cassette, and therefore, it may be unnecessary to remove the electrophoresis medium and manually apply the stain. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIG. 1  is a view of the top side of a 2-D PAGE device according to embodiments of the present invention; 
       FIG. 2  is a cross-sectional view of the 2-D PAGE device of  FIG. 1 ; 
       FIG. 3  is a cross-sectional view of the 2-D PAGE device of  FIG. 1  shown illustrating the flow channels for buffer solutions or staining reagents; 
       FIG. 4A–4C  is a cross-sectional view of a loading device according to embodiments of the present invention; and 
       FIG. 5  is a cross-sectional view of the 2-D PAGE device of  FIG. 1  shown with the loading device of  FIG. 3 . 
   

   DETAILED DESCRIPTION OF THE INVENTION 
   The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. This invention, however, should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. 
   In the drawings, the relative sizes of elements may be exaggerated for clarity. When an element is described as being on or adjacent another element, the element may be directly on or adjacent the other element, or other elements may be interposed therebetween. In contrast, when an element is referred to as being “directly on” another element, there are no intervening elements present. Like reference numerals in the drawings denote like members. 
   For ease of discussion, the exemplary embodiments disclosed herein may refer to IPG strip and polyacrylamide gel electrophoresis media. As would be appreciated by those of skill in the art, other electrophoresis media are interchangeable with IPG strips and polyacrylamide gels. Other acrylamide gels that may be used include gel media available from Invitrogen,™ Carlsbad, Calif. (U.S.A.) such as NuPAGE™ Bis-Tris (separation range 1.5 to 300 kDa), NuPAGE™ Tris-Acetate (separation range 30 to 400 kDa), Novex™ Tris-Glycine (separation range 6 to 500 kDa), Tricine (separation range 2 to 200 kDa), and Zymogram (separation range 30 to 200 kDa). 
   A 2-D PAGE device  10  is shown in  FIGS. 1 and 2 . The device  10  includes cassette housing  15 , which supports an IPG strip region  11  and a polyacrylamide gel region  13 . The cassette housing  15  can be about 8 cm long and about 8 cm wide. A sample solution can be introduced into the device  10  through a sample opening  35  and onto the IPG strip region  11 . IPG strips are commercially available under several trade names including BioRad™ from BioRad Laboritories, Hercules, Calif., U.S.A. 
   An IPG strip may be inserted into IPG strip region  11 . An IPG strip typically has a shelf life of about one year and should be kept dry and either refrigerated or frozen to increase shelf life. Therefore, it may be advantageous to store IPG strips separately from the cassette housing  10 , and to load and hydrate an IPG strip in the IPG strip region  11  just prior to performing electrophoresis. The IPG electrodes  25  and  27  can be used to apply the current to the IPG strip region  11  such that an IPG strip can perform isoelectric focusing to a sample added to an IPG strip through the sample opening  35 . 
   A protein sample may be loaded onto an IPG strip placed in the IPG strip region  11  using a “cup loading” method in which the sample is placed in a cup that interfaces with the hydrated IPG strip. Drip rods may also be used to load the sample as follows. Upon rehydration of the IPG strip, a protein sample may be placed into a drip rod that is then placed on the surface of the IPG strip. The drip rod can contact the IPG strip through the sample openings  35  or buffer openings  31 . The sample may be diluted, for example, by dissolving the sample in one or more of 9.5 M urea, 2–4% non-ionic or zwitterionic detergent, 1% Dithiothreitol (DTT), and 0.8% carrier ampholyte. See O&#39;Farrell PH (1975) “High resolution two-dimensional electrophoresis of proteins.” J Biol Chem 250: 4007–4021. Relatively hydrophobic proteins may be dissolved by a mixure of 2 M thiourea and 7 M urea instead of 9.5 M urea and/or other detergents. See Rabilloud T, Adessi, C, Giraudel A, Lunardi J (1997) “Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients.” Electro-phoresis 18: 307–316. 
   Isoelectric focusing typically requires exposing a sample solution to an IPG strip at about 15 degrees C. for about 12 hours at a constant voltage of about 300 V. Alternatively, various voltages may be applied depending on the IPG strip used and the proteins to be focused. For example, an eleven centimeter IPG strip can be focused for thirty minutes at 250 volts, sixty minutes of slow ramping to 8,000 volts, followed by fifteen to twenty kilovolt hours at 8,000 volts. 
   A polyacrylamide gel  47  ( FIG. 2 ), such as Tris-Glycine acrylamide, extends in a substantially flat planer two dimensional slab in the polyacrylamide gel region  13  ( FIG. 1 ). A removable dam  33  separates the IPG region  11  from the polyacrylamide gel region  13 . The removable dam  33  can be plexiglass. In operation, the removable dam  33  can be used to prevent spillage of a high salt content polyacrylamide gel  47  into the IPG strip region  11  while buffers can flush the IPG strip. Preferrably, the total salt concentration should not exceed 300 mM in a sample. The removable dam  33  can be inserted between the opposing plates  41  and  43  of the cassette housing  10  by cutting an incision in the top opposing plate  41 . A plexiglass removable dam  33  can be inserted in the incision and sealed with a high resistance vacuum sealant. The removable dam  33  can be removed prior to performing electrophoresis on the polyacrylamide gel  47 . After removal of the dam  33 , the incision can be sealed, for example, with sequencing tape. However, the removable dam  33  may not be necessary to separate the IPG strip region  11  from the polyacrylamide gel region  13 . For example, the cassette housing  10  may be placed at an angle to prevent a buffer from coming in contact with the polyacrylamide gel  47 . PAGE electrodes  17  and  19  in the form of metal filaments can be used to apply a voltage across the polyacrylamide gel region  13 . After proteins from a sample are focused on an IPG strip placed in the IPG strip region  11 , the protein from the IPG strip can be transferred from the IPG strip to the polyacrylamide gel region  13 . This transfer can be facilitated by a suitable buffer, which can be applied through the buffer openings  29  and  31 . As described above, an SDS/buffer can be used to increase the solubility and to impart a negative charge to the proteins in the solution. Other suitable buffers can be used such as dithiothreitol, tributyl phosphine, a mixture of 6M urea, 0.375 M Tris pH 8.8, 2% SDS, 20% glycerol, or 2% (w/v) Dithiothreitol (DTT), or a mixture of 6M urea, 0.375 M Tris pH 8.8, 2% SDS, 20% glycerol, or 2% (w/v) iodoacetamide. After adding a buffer through buffer openings  29  and  31 , a stopper such as a cylindrical piece of filter paper may be placed in the buffer openings  29  and  31 . Purified water can be added to the filter paper stopper so that the stopper can serve as a salt sink to remove impurities. 
   Once the dam  33  is removed and a buffer applied to the sample, an electric current is applied to the PAGE electrodes  17  and  19  so that the proteins that were isoelectrically focused on an IPG strip can travel towards the anode electrode  19 . As a result, the proteins in the sample are separated by their molecular weight in the polyacrylamide gel region  13 . Separation of the proteins in a sample by molecular weight along the polyacrylamide gel typically takes between about 30 and 40 minutes at ambient temperature. As understood by those of skill in the art, bromophenol Blue Dye can be used to determine the length exposure needed. 
   The IPG electrodes  25  and  27  can be connected to a current source (not shown) through buffer openings  29  and  31 , and PAGE electrodes  17  and  19  can be connected to current sources through electrode openings  21  and  23 . As will be appreciated by one of skill in the art, buffer openings  29  and  31 , electrode openings  21  and  23 , and sample openings  35  can be placed at other locations around housing  15 . For example, the sample opening  35  is preferably in the center of the IPG strip region  11 , but can also be situated off-center or at one end of the IPG strip region  11 . Although the IPG electrodes  25  and  27  should be placed at opposite ends of the IPG region  11 , additional buffer openings can be placed at various points along the IPG region  11 . The buffer openings  29  and  31 , electrode openings  21  and  23  and the sample opening  35  can be used to introduce various fluids into the cassette such as buffers, hydration solutions, sample solutions and staining reagents. These fluids may be introduced manually or automatically. 
   As can be seen in  FIG. 2 , the cassette housing  15  includes two opposing plates  41  and  43 . Spacers  45  separate opposing plates  41  and  43 . The polyacrylamide gel  47  forms a layer on one of the opposing plates  34 . A semi-permeable dialysis membrane  49  extends over top of the polyacrylamide gel  47 . The semi-permeable membrane  49  encloses and surrounds the polyacrylamide gel  47  to define a void  51  in between the membrane  49  and the other of the opposing plates  41 . The semi-permeable membrane can prevent the polyacrylamide gel  47  from expanding or moving into the void  51 . In certain embodiments, the polyacrylamide gel  47  and the membrane  49  can be chemically crosslinked together. 
   The void  51  that is defined by the membrane  49  can provide a space in which fluids may be introduced onto the polyacrylamide gel  47 . For example, as can be seen in  FIG. 3 , a staining reagent may be introduced by electrode openings  21  and  23 . The staining reagent can flow along arrows  63  across the membrane  49  and onto the polyacrylamide gel  47 . Staining reagents typically require about ninety minutes to stain a polyacrylamide gel. Therefore, embodiments of the present invention can provide staining inside the cassette and may eliminate the need to remove the polyacrylamide gel from the cassette housing  15  for staining. Alternatively, the polyacrylamide gel  47  can be removed from the housing  10  and subsequently stained. 
   Many of the steps described herein may be automated, reducing the need for skilled operators to perform the steps manually. For example, staining can be accomplished by automatically adding a staining reagent into the cassette such as with a machine configured to release the staining reagent into the electrode openings  21  and  23  at a predetermined time. As would be understood by those of skill in the art, the application of voltage on an IPG strip or polyacrylamide gel, the application of a buffer, the removal of the dam  33 , and the application of the staining reagent are examples of steps that may each be automated. For example, mechanical systems can be controlled by software and configured using known techniques to apply a solution through a specified opening in the cassette housing  15 , apply a voltage to a specified electrode, or remove the dam  33 , at predetermined times in order to perform 2-D PAGE. Automation of one or more of the steps may produce 2-D PAGE results with higher reproducibility and accuracy and require less intervention from a skilled operator. 
   The two opposing plates  41  and  43  and the spacers  45  can be formed of a single unitary member or, alternatively, from a plurality of parts. For example, the opposing plates  41  and  43  may be separately molded pieces that are joined by molded spacers  45 . Preferably the housing  15  is a plastic housing that is heat resistant. However, glass or other suitable materials may also be used. In certain embodiments, the spacers  45  can define an opening that is about 2 mm in height. 
   A loading device  110  that can be used to concentrate protein samples is shown in the  FIGS. 4A–4C  and  FIG. 5 . The loading device  110  includes a sample chamber  111  and two hydration chambers  113 A and  113 B adjacent the sample chamber  111 . The hydration chambers  113 A and  113 B are separated from the sample chamber  111  by semi-permeable membranes  117 A and  117 B. A sample solution  121  including proteins  123  can be placed in the sample chamber  111  through opening  127 . The sample chamber  111  includes a second opening  115 , which is closed when the sample  121  is placed in the sample chamber  111  in  FIG. 4B . 
   The hydration chambers  113 A and  113 B are filled with a hypertonic solution  119  in  FIG. 4B . The solution  119  is hypertonic with respect to the protein sample solution  121 . Therefore, turgor pressure increases in the hydration chambers  113 A and  113 B when the sample chamber  111  is filled with the protein solution  121 . As a result, electrolytes from the sample solution  121  flow into hydration chambers  113 A and  113 B by way of membranes  117 A and  117 B, and the protein sample solution  121  is concentrated as can be seen in  FIG. 4C . 
   As shown in  FIG. 4A–C , the opening  115  to the sample chamber  111  can be closed to allow concentration of the sample solution ( FIGS. 4A–B ), and subsequently opened such that the sample solution  121  can flow into an electrophoresis separation medium, such as an IPG strip  125  ( FIG. 4C ). As can be seen in  FIG. 5 , the loading device  110  can be used to load a sample solution  121  into the electrophoresis device  10 . The sample solution  123  follows arrows  61  onto IPG strip  63  through the opening  115  and the sample opening  35 . The loading device  110  can be used to provide automated loading of a concentrated solution into the electrophoresis device  10 . 
   In the drawings and specification, there have been disclosed typical illustrative embodiments of the invention and, although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation, the scope of the invention being set forth in the following claims.