Abstract:
High throughput electrophoresis having a plurality of samples migrating through a highly resistive separatory media under an electric field. Multiple electrophoresis runs are conducted using a single separatory media. Automated methods of data analysis yields quantitative and qualitative data using optical densitometer or other detection systems. Controlled operating temperatures and small geometry reduces dispersion inaccuracy in banded data.

Description:
RELATED APPLICATIONS  
       [0001]    This application claims priority to U.S. Provisional Application serial No. xx/yyy,yyy arising from conversion of non-provisional U.S. patent application Ser. No. 09/759,989, (entitled NANOPOROUS MEMBRANE REACTOR FOR MINIATURIZED REACTIONS AND ENHANCED REACTION KINETICS, and filed on Jan. 12, 2001), to a provisional application by petition filed in the U.S. Patent and Trademark Office on Jan. 4, 2002, the specification of which is herein incorporated by reference. 
     
    
     
       TECHNICAL FIELD  
         [0002]    This document relates generally to electrophoresis systems, devices and methods and particularly, but not by way of limitation, to an electrophoresis apparatus having high capacity and yielding high resolution data.  
         BACKGROUND  
         [0003]    Electrophoresis is an analytical tool for separating constituent elements of a sample. In particular, electrophoresis has been demonstrated to be effective for analysis of complex mixtures of molecules, such as proteins, peptides, amino acids, nucleic acids, inorganic ions, organic bases, organic acids, whole cells, deoxyribonucleic acid (DNA), and others. Electrophoresis can provide data concerning the mobility, size and charge of a molecule.  
           [0004]    In brief, electrophoresis entails moving an unknown sample through a porous medium. The various constituent elements migrate through the separation media at different rates depending upon their molecular properties, electrical charge to mass ratio and other factors. If the separation media is a gel, then one such factor is the size of the molecule. An external electric field, such as that provided by a DC power supply, may be applied to the separation media to promote migration of the sample.  
           [0005]    In one form of electrophoresis, the constituent elements appear as bands within the porous media, or separatory media. Typically, the separatory media, or gel, includes agarose or other gelatinous compounds. At high temperatures, agarose is a fluid and may be cast into a mold to create a porous media suitable for electrophoresis when cooled to room temperature.  
           [0006]    Various systems and techniques have been developed for detecting the bands created by the constituent elements within the separatory media. Examples of detection systems include measuring the electrical conductivity (or resistance) of each band, or measuring fluorescence or optical density based on light absorption of each band. An optical densitometer detection system includes a light source, often a laser, and optical detectors to measure emitted light. The quantitative output of the band detection system yields the identity of the constituent elements, and thus, the composition of the sample. Detectors may include electrochemical or radiochemical systems.  
           [0007]    Traditional electrophoresis is not renowned for high resolution or rapidity. Resolution is a measure of the quality of the separation of the bands and, in general, broad bands yields low resolution. The speed with which results can be achieved is based, in part, on the migration rate of the constituent elements. The field strength of an external electric field is limited by the physical dimensions of the separatory medium. If the electric field strength is too high, then the sample may be destroyed by excessive heat.  
           [0008]    Therefore, there is a need for an improved electrophoresis system and method that yields high resolution and rapid results.  
         SUMMARY  
         [0009]    The present subject matter is directed to apparatuses, systems and methods for performing analysis by electrophoresis. In one embodiment, the apparatus includes two fluid reservoirs coupled by a narrow chamber. The chamber includes parallel top and bottom plates and is filled with a separatory media. A void formed in one end of the medium located near one of the reservoirs is adapted to receive a comb-like membrane having approximately 100 teeth. One or more of the teeth of the membrane is spotted with a sample to be analyzed.  
           [0010]    In operation, the reservoirs contain a running buffer solution and an electric field is generated within the separatory medium by electrodes coupled to each of the reservoirs. An optical densitometer, or other band detector, provides graphical data concerning migration of the constituent elements of the samples. The graphical data includes one or more bands arranged in each of a plurality of discrete lanes where each lane is associated with a tooth of the membrane.  
           [0011]    In one embodiment, the apparatus is fabricated of glass, plastic, or other transparent material and the plates of the chamber are spaced apart by approximately 190 microns.  
           [0012]    The present subject matter also concerns a method of analyzing banded data arranged in a three dimensional grid system, in one embodiment. The three dimensions include, for example, an x-axis (spatial), a y-axis (time) and a z-axis (signal intensity). In a first step, each of a plurality of lanes are detected using a peak detection algorithm. The boundaries of each lane may be determined based on the dimensions of the membrane or by other means. In a second step, within each detected lane, each of a plurality of bands are detected using a peak detection algorithm. The peak detection algorithm may operate on data from the optical densitometer or other detector.  
           [0013]    In one embodiment, the separatory medium includes a gel formed within the chamber by heating and casting in situ. In one embodiment, the molten gel is poured into a reservoir and forced into the chamber by means of air pressure. A temporary spacer element may be inserted into the chamber to form a void to receive the membrane.  
           [0014]    In one embodiment, automated methods are used for spotting the teeth of the membrane, inserting the membrane and applying the fluids to perform electrophoresis.  
           [0015]    Other aspects of the invention will be apparent on reading the following detailed description of the invention and viewing the drawings that form a part thereof. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0016]    In the drawings, like numerals describe substantially similar components throughout the several views. Like numerals having different letter suffixes represent different instances of substantially similar components.  
         [0017]    [0017]FIG. 1 is a sectional view of one embodiment of an electrophoresis apparatus.  
         [0018]    [0018]FIG. 2 is a perspective view of one embodiment of an electrophoresis apparatus.  
         [0019]    [0019]FIGS. 3A and 3B illustrate one embodiment of a membrane.  
         [0020]    [0020]FIGS. 4A and 4B are sectional views of a portion of a membrane.  
         [0021]    [0021]FIG. 5 is a view of a portion of an electrophoresis apparatus.  
         [0022]    [0022]FIG. 6 is a sectional view of a portion of one embodiment of an electrophoresis apparatus.  
         [0023]    [0023]FIG. 7 is a sectional view of a portion of one embodiment of an electrophoresis apparatus.  
         [0024]    [0024]FIG. 8 is a view of a spacer in accordance with one embodiment of the present subject matter.  
         [0025]    [0025]FIG. 9 is a perspective view of a tool for use with one embodiment of an electrophoresis apparatus.  
         [0026]    [0026]FIG. 10 is a perspective view of a tool coupled to a partially assembled electrophoresis apparatus.  
         [0027]    [0027]FIG. 11 is a perspective view of an electrode assembly for use with one embodiment of an electrophoresis apparatus.  
         [0028]    [0028]FIG. 12 illustrates a flow chart of a method for preparing and operating an electrophoresis apparatus.  
         [0029]    [0029]FIG. 13A is a photograph of a screen displaying data derived from an electrophoresis apparatus according to the present subject matter.  
         [0030]    [0030]FIG. 13B illustrates selected details of the data shown in FIG. 13A.  
         [0031]    [0031]FIG. 14 illustrates a flow chart of a method for analyzing banded data from an electrophoresis apparatus.  
         [0032]    [0032]FIG. 15 illustrates a flow chart of a method for analyzing banded data from an electrophoresis apparatus. 
     
    
     DETAILED DESCRIPTION  
       [0033]    In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration specific embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that the embodiments may be combined, or that other embodiments may be utilized and that structural, logical and electrical changes may be made without departing from the spirit and scope of the present invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims and their equivalents.  
         [0034]    By way of overview, the present system includes a pair of reservoirs coupled by a narrow chamber. Within the narrow chamber is a separatory media, or a gel in one embodiment. Samples to be analyzed are spotted onto the teeth of a comb-like membrane having approximately 100 teeth. The membrane is inserted into a void formed in the separatory medium and running buffer solutions are introduced into the reservoirs. The running buffer solutions provide pH buffering and conducts an electric field through the separatory medium.  
         [0035]    The constituent elements of each sample migrate away from the membrane and through the separatory medium based on various factors including the resistance through the medium, the electric field strength and the size, electric charge and configuration of each constituent element. Data concerning the migration of the constituent elements can be determined by optical densitometer or other band detection methods.  
         [0036]    In one embodiment, the resulting bands are analyzed using a method which yields quantitative data. In one embodiment, the resulting bands are analyzed using a method which yields qualitative data. Each tooth of the membrane bearing a sample forms a discrete lane within the separatory medium. Each lane may have one or more bands corresponding to each of the constituent elements of the sample on that particular tooth of the membrane. One method entails, first, detecting the lanes, and second, detecting bands within each lane. In one embodiment, lanes are detected by summing the data points along a first axis, normalizing the data and determining the peaks using a peak detection algorithm. Within each detected lane, the process is repeated to detect bands. In particular, band detection includes summing the data points along a second axis and determining the peaks using a peak detection algorithm.  
         [0037]    The membrane described herein is an example of a sample delivery device. Other sample delivery devices are also contemplated, including manual devices for providing a sample to the separatory medium of the present subject matter.  
         [0038]    The present subject matter is adapted for large volume analysis with high resolution output. It is believed that the narrow chamber dimensions provides a uniform environment for migration of the constituent elements of the samples. The chamber also presents a high resistance media for the constituent elements and thus, reduces temperature gradations. The present subject matter allows application of a high intensity electric field thus yielding high resolution data with rapid migration.  
         [0039]    Structure  
         [0040]    [0040]FIG. 1 illustrates a sectional view of one embodiment of system  100  for conducting electrophoresis analysis in accordance with one embodiment of the present subject matter. Not shown in the figure are end caps for the reservoirs and the chamber and other structure.  
         [0041]    In one embodiment, device  200  includes reservoir  210  and reservoir  220 . Reservoirs  210  and  220  are coupled by a chamber which is bounded by upper plate  250  and lower plate  240 . The chamber contains gel, or other separatory media,  230  and at least a portion of membrane  280 . Reservoir  210  is capped with cover  322  and reservoir  220  is capped with cover  312 . In one embodiment, cover  322  is coupled to anode  325  and cover  312  is coupled to cathode  315 . In one embodiment, cover  322  is coupled to cathode  315  and cover  312  is coupled to anode  325 . Anode  325  and cathode  315  are coupled to DC power supply  300  by leads  318  and  308 , respectively. An electrical connection between anode  325  and lead  318  is established by connector  320 . An electrical connection between cathode  315  and lead  308  is established by connector  310 . It will be noted that anode  325  and cathode  315  may be positioned in a manner separately from covers  322  and  312 , respectively. For example, anode  325  and cathode  315  may include flat conductors positioned within the respective reservoirs and bonded to lower plate  240 .  
         [0042]    In one embodiment, detection system  340  includes an optical detector  330  coupled to an optical densitometer  332 . Densitometer  332  is coupled to processor  350 . Other embodiments are also contemplated, such as for example, a fluorescence detection system. Processor  350 , in one embodiment, provides data analysis as described elsewhere in this document.  
         [0043]    Device  200  may be fabricated of translucent or opaque material including plastic, glass or other materials. In one embodiment, either upper plate  250  or lower plate  240  or both are fabricated of material compatible with detection system  340 . In one embodiment, reservoir  210 , reservoir  230 , upper plate  250  and lower plate  240  are separately fabricated and bonded together in the manner illustrated to form leakproof joints.  
         [0044]    In one embodiment, cover  322  and cover  312  protect the contents of reservoirs  210  and  220  from evaporation, foreign objects, and other contaminants. Covers  322  and  312  need not fit reservoirs  210  and  220 , respectively, with a fluid tight seal.  
         [0045]    In one embodiment, cover  322  and cover  312  carry electrical terminals. For example, anode  325  is coupled to cover  322  and cathode  315  is coupled to cover  312 . With cover  322  in position on reservoir  210 , anode  325  is in electrical contact with running buffer  215  and with cover  312  in position on reservoir  220 , cathode  315  is in electrical contact with running buffer  225 .  
         [0046]    Separatory medium  230  occupies the chamber between upper plate  250  and lower plate  240 . In one embodiment, upper plate  250  and lower plate  240  are substantially parallel and spaced apart by a distance of  190  microns. Distances greater than or less than  190  microns are also contemplated. In one embodiment, separatory medium  230  is formed in place by heating to a melting point and forcing into the space between upper plate  250  and lower plate  240 .  
         [0047]    Separatory medium  230  may include agarose or a gel known to one of skill in the art. In one embodiment, the separatory medium is pure agarose (in the range of, for example, 0.05-5% or 0.01 to 30%). In one embodiment, the separatory medium includes a composite. An example of a composite includes agarose and linear polyacrylamide (LPA) gel (with agarose in the range of, for example, 0.05-5%, or 0.01 to 30% and LPA in the range of, for example, 0.05-10%). In one embodiment, the separatory media is adapted to separate molecules in various molecular weight ranges. In various embodiments, the weight range is between 1,000 to 10,000,000 or 10,000 to 1,000,000,000 although other weight ranges are also contemplated.  
         [0048]    Resolution of electrophoresis data is believed to be affected by temperature gradations across the separatory medium and the strength of the electric field through the medium. Here, the narrow dimensions of the chamber yields lower temperature variability, and thus, sharper bands of data with higher resolution. Higher temperatures cause the samples to move more rapidly, leading to more regularly shaped bands and less dispersion or blurred images. Thus, with less temperature gradients, the resolution improves since the bands remain sharp and narrow.  
         [0049]    In addition, it is believed that the narrow dimensions of the chamber permit the use of a greater external electric field strength. For example, in one embodiment, the external voltage provided by supply  300  may be in the range of 50 to 100 volts per cm, with a typical value of 75 volts per cm. Thus, over a 10 cm distance, the supply voltage may be 1,000 volts. External cooling of device  200  may allow voltages greater than 100 volts per cm. For example, liquid cooling provided to upper plate  250  or lower plate  240  may allow use of a greater external field, and thus, higher resistance pathways and therefore, yield higher throughput. Liquid cooling may include water cooling or circulating a cooling agent or fluid in the proximity of the separatory medium. When using approximately 75 volts per cm to provide an external field, the run time for system  100  is typically 5 to 25 minutes in duration.  
         [0050]    Electro osmotic forces (EOF) arising from application of the external electromagnetic field, may tend to move separatory medium  230  from within the chamber. In one embodiment, separatory medium  230  includes an additive that dynamically coats the interior surface of the chamber, thereby attenuating such electro osmotic forces. One such additive includes a polymer, such as polyvinylpiperidone (PVP), or dimethylacrylamide or other hydrophilic linear polymer.  
         [0051]    In the absence of a coating on the interior of the chamber, the electrostatic charge of the fluid is generally positive fluid and the charge of the surface of the chamber is generally negative. Using a polymer coating yields a neutral surface and a neutral fluid, thus reducing electro osmotic forces.  
         [0052]    Void  260  is formed in one end of separatory medium  230  located near reservoir  220  and at least partially within the space between upper plate  250  and lower plate  240 . Void  260  is adapted to accept membrane  280  and a quantity of focusing water  270 .  
         [0053]    The constituent elements of the samples delivered to separatory medium  230  by membrane  280  travel in the direction indicated by arrow  282 . The constituent elements are repelled by the cathode  315  end and attracted to anode  325  end.  
         [0054]    [0054]FIG. 2 illustrates a perspective view of one embodiment of device  200  with cover  312  and cover  322  removed for the sake of clarity. In the embodiment illustrated, reservoir  210  and reservoir  220  are machined from a solid plastic block. Reservoir  210  and reservoir  220  are coupled to upper plate  250  and lower plate  240 .  
         [0055]    In the figure, optical densitometer detector  330  is positioned at a near end of separatory medium  230 . Detector  330  is mounted on a track mechanism (not shown) and travels in the directions indicated by the arrowheads on line segment  362 . In one embodiment, detector  330  includes a central optical fiber for transmitting a column of light and a plurality of sensor fibers distributed about the circumference of the central optical fiber for receiving reflected light. In cycling back and forth, detector  330  provides a signal based on the intensity of the bands generated by the constituent elements as each migrates through separatory medium  230 . The output of detector  330  is processed by detector system  332  of FIG. 1 and further processed by processor  350 , also of FIG. 1. In one embodiment, device  200  is mounted to a commercial stage apparatus and detector  330  is powered by a motorized carriage mechanism coupled to the stage.  
         [0056]    [0056]FIGS. 3A and 3B illustrate views of membrane  280 . In FIG. 3A, membrane  280  includes comb  290  and reinforcer  285 . Reinforcer  285  includes alignment notches  295  at each end.  
         [0057]    [0057]FIG. 3B illustrates a close view of membrane  280 . Comb  290  includes a plurality of teeth, some of which are visible in the view shown and are denoted herein as X, X−1, X−2, X−3. In various embodiments, X is 96 or 100. In one embodiment X is an integer between 50 and 150. Comb  290  is bonded to reinforcer  285 , forming a laminate structure having visible joint line  292 . Alignment notch  295  is visible in FIG. 3B.  
         [0058]    In one embodiment of system  100 , membrane  280  is installed in device  200  in such a manner that the thicker portion of comb  290  and reinforcer  285  is positioned under reservoir  220  and a portion of the teeth of comb  290  are inserted within the space between upper plate  250  and lower plate  240 . Other embodiments are also contemplated, such as for example, insertion of membrane  280  to a greater or lesser depth.  
         [0059]    In one embodiment, membrane  280  includes a porous or nanoporous structure. Methods, materials, devices and systems concerning nanoporous membranes are described in U.S. patent application Ser. No. aa/bbb,bbb, entitled NANOPOROUS MEMBRANE REACTOR FOR MINIATURIZED REACTIONS AND ENHANCED REACTION KINETICS, filed Jan. 14, 2002, invented by Andras Guttman, Zsolt Ronai, and Csaba Barta and assigned to Syngenta Participation AG, the specification of which is incorporated herein by reference.  
         [0060]    [0060]FIG. 4A illustrates a perspective view of a portion of membrane  280  when viewed in the direction of cut-line A-A shown in FIG. 3A. The bond line between comb  290  and reinforcer  285  and joint line  292  are visible in the figure. In FIG. 4A, teeth X−78, X−77, X−76 and X−75 of comb  290  are shown as rectangular teeth, however, it will be appreciated that other shapes and configurations are also contemplated. For example, the teeth of comb  290  may have a circular cross-section or include rounded ends in contrast to the square cuts shown. It will be appreciated that comb  290  is illustrated as having teeth of uniform dimensions and features, however, alternative configurations are also contemplated. For example, the teeth may have progressively shorter dimensions or have larger area or include triangular, tapered, or pyramid shaped teeth.  
         [0061]    [0061]FIG. 4B illustrates a perspective view of representative tooth X−78 of comb  290  coupled to a portion of reinforcer  285 . The sample to be analyzed by electrophoresis may be deposited on one or more of surfaces  290 A,  290 B and  290 C. In one embodiment, the sample is deposited onto membrane  280  robotically. On any particular comb  290 , each tooth may carry a unique sample or some teeth may carry the same sample.  
         [0062]    [0062]FIG. 5 illustrates a top view of device  200  with cover  322  and cover  312  removed and before installation of separatory medium  230  and membrane  280 . Visible in the figure are the boundary walls of reservoir  210  and reservoir  220  coupled by top plate  250 .  
         [0063]    [0063]FIG. 6 illustrates a view of a portion of membrane  280  having comb  290  and reinforcer  285 . Upper plate  250  and lower plate  240  are shown to be of substantially the same thickness dimension and are spaced apart, in one embodiment, at a distance of 190 microns. In one embodiment, upper plate  250  and lower plate  240  may be of different thickness dimensions with one greater than the other. In one embodiment, the combined thickness of laminated structure  280 , including comb  290  and reinforcer  285 , is approximately 130 microns and the length of the portion of comb  290  inserted into the chamber between upper plate  250  and lower plate  240  is approximately 1 to 2 mm.  
         [0064]    To improve the accuracy of data generated by device  200 , it is preferable that upper plate  250  and lower plate  240  are substantially parallel. If the space between upper plate  250  and lower plate  240  is too small, then insertion of membrane  280  may be precluded. In addition, the resistance of the separatory medium  230  will vary with location and thus, the current flow through separatory medium  230  may be irregular. Irregular current flow leads to different speeds of constituent element migration, thus impairing the value of the data generated.  
         [0065]    Maintaining uniform and accurate separation of the plates  250  and  240  over the width of the chamber (which, in one embodiment is a distance of 12 to 25 cm) may be accomplished using any one of, or a combination of, techniques. For example, at the time of manufacturing of device  200 , a suitable temporary spacer may be inserted between the lower plate  240  and upper plate  250  before bonding of reservoirs  210  and  220  to plates  240  and  250 . After the bonds have cured, the temporary spacer is removed and device  200  is ready for installation of the separatory medium. In one embodiment illustrated in FIG. 7, the thickness dimension of upper plate  250  is increased relative to that of lower plate  240 .  
         [0066]    The dimensional stability of the upper plate  250  relative to lower plate  240  is derived from external means. For example, stability may be provided by the structural strength of reservoirs  210  and  220  or by the bond between reservoirs  210  and  220  and the upper plate  250  and lower plate  240 .  
         [0067]    Method  
         [0068]    As noted above, separatory medium  230  preferably presents a uniform resistance to migration of the constituent elements of the sample. The following discussion concerns a method for installing the separatory medium into the chamber between upper plate  250  and lower plate  240 .  
         [0069]    In brief, one embodiment provides that the separatory medium is heated to more easily flow into the narrow chamber, poured into either reservoir  210  or reservoir  220  and pumped through the chamber under pneumatic pressure. Void  260  is prepared by inserting a suitable spacer into separatory medium  230  while still molten.  
         [0070]    [0070]FIG. 8 illustrates a portion of spacer  360  that may be used for preparing void  260  in separatory medium  230 . In one embodiment, dimension Z is approximately 150 microns and dimension Y is approximately 3 to 5 mm and width dimension W is approximately 15 cm, however, other dimensions are also contemplated. Dimensions Z, Y and W are selected to provide a suitable space for insertion of membrane  280  into separatory medium  230 . Spacer  360  may be fabricated of plastic, metal or other suitable material. In one embodiment, spacer  360  is made of plastic shim stock and includes a thicker portion to allow easy manipulation for insertion and removal.  
         [0071]    [0071]FIG. 9 illustrates apparatus  405  that may be used to apply pneumatic pressure to a molten separatory medium  230 . In one embodiment, apparatus  405  includes gasket  410  bonded to rigid channel  400 . Channel  400  may include an extruded or formed aluminum or plastic structure. Gasket  410  may include a rubberized surface of approximately 0.5 cm thickness. Tube  420  is coupled at a first end to fitting  440  and at a second end  422  to a source of pressurized air. Fitting  440  may be threadably engaged with channel  400  and includes port  450  which communicates pressurized air to manifold  430  along the length of channel  400 . Apparatus  405  may be fabricated using other structures and materials.  
         [0072]    In one embodiment, separatory medium  230  may be moved into position by application of a vacuum source. Apparatus  405  may be coupled to a vacuum source and coupled to device  200  in a manner that draws separatory medium  230  into the chamber.  
         [0073]    [0073]FIG. 10 illustrates apparatus  405  mounted on device  200 . Apparatus  405  is positioned with gasket  410  in contact with reservoir  220 . Gasket  410  is compressed between channel  400  and reservoir  220  by a clamping force. In one embodiment, apparatus  405  may be urged in the direction of arrow  460  by a pair of fixture clamps attached to a surface on which device  200  is placed. Thus, device  200  and apparatus  405  are urged in the directions shown by arrows  455  and  460 , respectively.  
         [0074]    Air pressure introduced into flexible tube  420  is distributed through port  450  and manifold  430  to reservoir  220 . Molten separatory medium  230  in reservoir  220  is pneumatically forced into the space between upper plate  250  and lower plate  240 .  
         [0075]    [0075]FIG. 11 illustrates cover  322 . Cover  322  serves as a lid for reservoir  210  and carries anode  325 . Cover  322  includes standoff  328  and electrical conductor  325 , mounting block  321  and terminal  320  coupled to electrical lead  318 . Cover  322  and standoff  328  may be fabricated of insulative material such as plastic, glass or other suitable material. Cover  322  and standoff  328  are bonded together. Terminal  320  provides an electrical connection between lead  318  and conductor  325 . In the embodiment shown, conductor  325  is a bare wire laced through a hole in cover  322  and along the length of standoff  328 .  
         [0076]    Preparation  
         [0077]    [0077]FIG. 12 illustrates flowchart  500  for preparing and conducting electrophoresis analysis using one embodiment of apparatus  200 .  
         [0078]    The method begins at  510 , and at  515 , device  200  is heated to a temperature of approximately 60° C. At  525 , separatory medium  230  is heated to a molten state at a temperature of approximately 60° C. In one embodiment, separatory medium  230  is viscous fluid at approximately 60° C. and relatively solid at 32° C.  
         [0079]    At  530 , separatory medium  230  is poured into reservoir  220 . At  535 , pneumatic pressure is applied to reservoir  220  using apparatus  405  as illustrated in FIGS. 9 and 10. Pneumatic pressure may be provided by a regulated source of air pressure such as air compressor. At  540 , migration of separatory medium  230  may be verified by visually observing a contiguous line of separatory medium  230  emerging from reservoir  210 . After verifying that separatory medium  230  has migrated through the space between upper plate  250  and lower plate  240 , apparatus  405  may be removed from device  200 . In one embodiment, device  200  is positioned with a slight incline from horizontal, thus bringing gravity to bear on the migration of separatory medium  230 .  
         [0080]    At  545  and at a time while separatory medium  230  is at an elevated temperature, temporary spacer  360  may be inserted into separatory medium  230  accessible through reservoir  220 . In one embodiment, spacer  360  penetrates into the separatory medium for a depth of approximately 3 to 5 mm.  
         [0081]    At  550 , system  100 , including device  200  and separatory medium  230 , is allowed to cool to room temperature. Cooling may take approximately five to ten minutes and may be accelerated by refrigeration. In one embodiment, system  100  is cooled to a temperature where separatory medium  230  is no longer molten.  
         [0082]    At  555 , spacer  360  is removed from separatory medium  230 , thus forming void  260 . In one embodiment, void  260  provides a depth stop for the insertion of membrane  280 .  
         [0083]    The foregoing procedure describes one example of a method for making a separatory medium suitable for use with system  100 . Other procedures, or sequences of steps may be implemented.  
         [0084]    Separatory medium  230  formed in the manner described above may be used for multiple electrophoresis runs. For example, in one embodiment, a single separatory medium  230  may be used for all runs that may be completed in a day or other period of time.  
         [0085]    Consider next the procedure for analyzing samples on a membrane. At  560 , a small quantity of focusing water is injected into void  260 . Ordinary water, having a very high resistance, is used for focusing water. Focusing water promotes an intense localized electric field, thus the bands tend to focus more rapidly.  
         [0086]    At  565 , membrane  280 , having been previously treated with a sample to be analyzed, is inserted into separatory medium  230 . Insertion may be accomplished manually or by robotic means, and in one embodiment, includes passing membrane  280  through reservoir  220 . Membrane  280  is inserted with the teeth of comb  290  positioned proximate separatory medium  230  and reinforcer  285  distal from separatory medium  230 .  
         [0087]    At  570 , running buffer  215  is introduced into reservoir  2   10  and running buffer  225  is introduced into reservoir  220 . Running buffer  215  and running buffer  225  may be of the same or different composition. Running buffers  215  and  225  cover the exposed portions of separatory medium  230  and have a level sufficient to establish electrical contact with anode  325  and cathode  315 , respectively.  
         [0088]    At  575 , anode  325  and cathode  315  are positioned to contact running buffers  215  and  225 , respectively. In system  100  illustrated in FIG. 1, this includes placing covers  322  and  312  in position atop reservoirs  210  and  220 , respectively.  
         [0089]    At  580 , an electrical potential is applied across anode  325  and cathode  315  from direct current (DC) power supply  300 . Supply  300  may include a regulated voltage supply or a battery.  
         [0090]    At  585 , lanes and bands are detected within separatory medium  230 . The bands may be detected using apparatus as shown in FIG. 1 or FIG. 2. In various embodiments, detecting bands may entail using a UV/Vis detector, a fluorescence detector, a conductivity detector, an electrochemical detector, a mass spectrometer, a radioactive detector, a post column reaction detector, an optical densitometer, or other types of detectors.  
         [0091]    The procedure ends at  590 .  
         [0092]    Data Analysis  
         [0093]    [0093]FIG. 13A depicts a photograph of a screen while displaying data derived from an electrophoresis apparatus according to the present subject matter. The photograph illustrates six lanes with each lane having a plurality of bands. In the photograph, the bands have migrated in a direction from bottom to top. In one embodiment, data is derived from more than six lanes, however, for purposes of clarity, only six lanes are depicted in the photograph.  
         [0094]    [0094]FIG. 13B illustrates selected details of the data shown in FIG. 13A. The figure shows a screen shot of data derived from system  100  in one embodiment. Each lane, or column, of data appearing in the figure, herein denoted as X−5, X−4, X−3, X−2, X−1 and X, corresponds to a particular tooth, or lane, in comb  290 . Since the present system is suitable for use with many samples, it will be appreciated that comb  290  may have many separate teeth, or lanes, and each lane corresponds to a sample arranged in the image in the form of a column. Thus, in one embodiment, a particular electrophoresis run may generate 100 lanes of data.  
         [0095]    The bands appearing in the figure may be generated by an optical densitometer or other detection system.  
         [0096]    In particular, consider column X−5. Within column X−5, eleven bands, or blots, appear. Each band denotes a particular constituent element of the sample carried by tooth X−5. With the possible number of lanes in excess of  100 , analysis of even a single run may entail analysis of  1100  individual bands. It is believed that the present system may enhance reliability and accuracy of the analysis of such a large amounts of data.  
         [0097]    The screen shot appearing in the figure indicates spatial data on the horizontal, or x-axis, denoted as axis  610 , and time on the vertical, or y-axis, denoted as axis  605 . A z-axis can be construed to represent the intensity of the signal. Data generated by the detection system  340  yields the individual bands. Representative bands appearing in the figure within column X−5 are denoted as  635 A,  635 B,  635 C,  635 C and  653 D.  
         [0098]    Detection system  340  is configured to acquire data at a rate sufficient to scan 100 lanes. For example, in one embodiment, the sampling rate of detection system  340  is approximately 2 kHz. In one embodiment, an external clock provides synchronization between data acquisition and movement of the scanning detector.  
         [0099]    The picture in the figure represents a single line in the separatory medium (for example, at a position 10 cm from the injection side) plotted in time. Bands that migrate through the separatory medium fast will reach this line/detector, the earliest and are the first, or lowest, bands in the picture. Slower bands reach the detector later and thus, appear higher in the picture.  
         [0100]    [0100]FIG. 14 illustrates one embodiment of method  650  for analyzing data. Beginning at  655 , the method includes discerning the individual lanes from among a field of lanes, as at  660 . At  665 , and within each lane, the method also includes discerning individual bands. The method ends at  670 .  
         [0101]    [0101]FIG. 15 illustrates method  700  for analyzing data and begins at  705 . To discern each individual lane, the method entails viewing the data as a two-dimensional picture. At  710 , data points are summed along axis  610 , that is between the bottom and the top of the image in FIG. 13B. For example, in column X−5, the data represented by each of the eleven bands are summed along the time axis. At  715 , the data is normalized to reduce the range of data. At  720 , and using the normalized data, a peak detection algorithm is used to identify individual lanes. In column X−5, for example, the peak occurs at point  615  and vertical dotted line  625  denotes the peak. At  725 , and continuing with the example of column X−5, left boundary  645  and right boundary  640  on either side of the peak  615  (and line  625 ) are then constructed. In one embodiment, the position of boundaries  645  and  640  may be determined based on the physical dimensions of comb  290 .  
         [0102]    Having determined the boundaries for each lane, at  735 , the data points are summed along axis  610 . Then, data points are summed between left boundary  645  and right boundary  640 . At  745 , using a peak detection algorithm, each band is identified with a particular peak value. At  750 , it is presumed that the peak ordinarily occurs at the most intense portion of each band, thus yielding the position for that particular band. However, the time value may be marked at either edge of the band or at another location relative to the band. In one embodiment, the peak, shown by vertical dotted line  625 , is displaced to one side or the other. Further analysis can proceed using the center, peak, or any other location within the boundaries of the lane. In column X−5 of the figure, the eleven bands, some of which are denoted as  635 A,  635 B,  635 C and  635 D have values marked by lines  630 A,  630 B,  630 C and  630 D, respectively. The method ends at  755 .  
         [0103]    In one embodiment of the present subject matter, the methods described herein are implemented in executable computer software. For example, a personal computer coupled to a detector, such as an optical detector, is configured to execute programming to identify the lanes and to identify the bands within each lane. The software can be adapted to execute on a computer and stored on removable storage media.  
         [0104]    Alternative Embodiments  
         [0105]    In one embodiment, automation systems may be used to further increase throughput of samples. For example, an automatic membrane insertion tool may be used to robotically place membrane  280  in the chamber. Furthermore, robotic spotting equipment may place samples on the teeth of membrane  280 . Robotic fluid dispensing equipment may be used to inject focusing water  270  or place running buffers  215  and  225  in their respective reservoirs. Timers or other robotic equipment may also be used to cycle power supply  300  to predetermined voltage levels. In one embodiment, an automated process may perform tens or hundreds of electrophoresis runs without human intervention.  
         [0106]    Conclusion  
         [0107]    The above-described system provides, among other things, a system, apparatus and method for performing electrophoresis with high resolution and high throughput while using a smaller quantity of chemicals.  
         [0108]    It will be appreciated that the methods described herein may be performed in different orders than described and that portions of a method may be repeated.  
         [0109]    It is to be understood that the above description is intended to be illustrative, and not restrictive. Many other embodiments will be apparent to those of skill in the art upon reviewing the above description. The scope of the invention should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.