Abstract:
An automated assembly for performing first dimension electrophoresis is described herein that includes a supply magazine, an electrophoresis tank and an automated transferring device that robotically transfers biological samples from sample vials retained in the supply magazine, and delivers the biological samples one by one to tube gels supported in a rack within the electrophoresis tank. The transferring device is configured to move in three dimensions with respect to the supply magazine and the rack for flexible sample delivery.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application is a Continuation-In-Part application of U.S. application Ser. No. 09/621,484 filed Jul. 21, 2000 now U.S. Pat. No. 6,537,434 for First Dimension Electrophoresis Separation Method and Apparatus, which hereby incorporated by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention is directed to a method and automated apparatus for performing isoelectric focusing of macromolecules, and particularly proteins. More particularly, the present invention is directed to an automated apparatus for supplying protein samples from a sample well to a gel tube for the first dimension isoelectric focusing of the protein sample. 
     BACKGROUND OF THE INVENTION 
     Isoelectric focusing (IEF) is an electrophoretic technique for the analysis, separation and purification of various biological materials. Since many of the complex molecules of biological interest are amphoteric in nature, they are typically amenable to IEF separation. 
     Isoelectric separation is a known process that has been used for many years. An isoelectric focusing gel, such as an acrylamide gel, is placed or polymerized in a tube having open ends. Each open end is positioned in a bath containing a buffer solution. One buffer solution is typically a sodium hydroxide solution to contact one end of the gel tube. The other buffer solution is typically a phosphoric acid solution at the opposite end of the tube to produce a pH gradient between the two ends of the tube. When current is applied, the two buffer solutions, together with ampholytes incorporated into the gel composition or titratable gel monomers incorporated into the gel, provide an electric potential through the gel along the length of the tube. The sample to be analyzed is applied to a top end of the gel in a tube and an electric current is applied to an electrode in each of the buffer solutions. The molecules in the sample migrate through the gel under the influence of the electric potential until they reach their isoelectric point. 
     The separation of macromolecules, and particularly proteins, often is carried out by a two-dimensional electrophoresis separation process. The two-dimensional electrophoresis separation typically involves the sequential separation by isoelectric focusing of a sample in a gel tube followed by slab gel electrophoresis. The isoelectric focusing process is often referred to as first dimension separation. Slab gel electrophoresis, often referred to as second dimension separation, utilizes an electrophoresis gel molded between two glass plates. A gel strip or cylinder in which the protein sample has been resolved by the first dimension isoelectric focusing is placed along one edge of the slab gel. The opposite ends of the gel slab are immersed in a buffer solution and an electric current is applied between the ends to provide an electric potential through the gel slab. The proteins are then allowed to migrate through the gel slab under an applied voltage. 
     Charged detergents, such as sodium dodecyl sulfate, contained in the slab gel bind to the protein molecules. The detergents tend to unfold the protein molecules into rods having a length proportional to the length of the polypeptide chain and thus proportional to the molecular weight of the polypeptide. A protein complexed with a charged detergent is highly charged, which causes the protein-detergent complex to move in an applied electric field. When the slab gel, such as a polyacrylamide gel, functions as a sieve, the movement of the longer and higher molecular weight molecules is retarded compared to the shorter, lower molecular weight molecules. 
     Electrophoresis separation is generally labor intensive since numerous samples are run simultaneously. In the first dimension separation, the gel tubes are prepared and placed in a suitable tank of buffer solutions. The protein samples are then manually placed on the end of a gel tube. When hundreds of protein samples are prepared daily for isoelectric focusing, the manual steps significantly increase the time requirements for performing the first dimension separation. Accordingly, there is a need in the industry for improved methods and devices for conducting first dimension isoelectric focusing. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to a method and apparatus for the electrophoresis separation of macromolecules and particularly proteins. More particularly, the invention is directed to an automated apparatus for first dimensional isoelectric focusing of proteins and other macromolecules. 
     Accordingly, a primary aspect of the invention is to provide an automated apparatus for handling and manipulating a large number of samples for electrophoresis separation. 
     Another aspect of the invention is to provide an automated apparatus for sequentially transferring a large number of biological samples from a respective sample container to a respective gel tube for performing electrophoresis separation of the sample. 
     A further aspect of the invention is to provide an automated apparatus for transferring a biological sample from a sample container to a gel tube where information identifying the sample and the location of the sample is stored in a computer. 
     Another aspect of the invention is to provide an automated apparatus for electrophoresis separation including a sample container magazine having a holding device for holding a sample container stationary while a sample is being removed. 
     A further aspect of the invention is to provide an automated apparatus for electrophoresis separation including a computer controlled arm having a pipette for piercing a septum in a sample container and removing a selected quantity of a sample from the container. 
     Still another aspect of the invention is to provide an automated apparatus for electrophoresis separation including a computer controlled arm having a pipette, and a sample container holding device for holding the sample container stationary while the pipette penetrates and is withdrawn from a septum in the sample container. 
     Another aspect of the invention is to provide an automated apparatus for transferring a plurality of biological samples to a respective gel tube where the assembly has a computer for recording and tracking the location of the samples. 
     A further aspect of the invention is to provide an automated apparatus for transferring a plurality of samples to a respective gel tube, wherein the apparatus includes a support member, a movable arm coupled to the support member and is movable along a longitudinal dimension of the support member, and a pipette mounted on the movable arm that is movable vertically for withdrawing a sample from a container and for dispensing a sample to a gel tube. 
     Another aspect of the invention is to provide an automated apparatus for electrophoresis separation having a robotic arm with a pipette that is movable in three dimensions and where the pipette is movable from a sample withdrawing position to a sample dispensing position. 
     A further aspect of the invention is to provide an automated apparatus for electrophoresis separation of macromolecules, where the apparatus has a plurality of electrophoresis gel tanks, each supporting a parallel row of gel tubes. The apparatus has a movable robotic arm that is able to transfer a sample from a sample vessel to a selected gel tube. 
     Another aspect of the invention is to provide a rack for supporting a plurality of gel tubes in an electrophoresis tank and where the rack has an open well containing a buffer solution for electrophoresis separation and a guide for guiding a pipette to an end of a gel tube that is positioned in the bottom of the well. 
     Still another aspect of the invention is to provide an automated transferring device for transferring samples from a sample container to a gel tube where the device includes a stationary cover member positioned above an electrophoresis tank and where the cover member includes a plurality of apertures aligned with the gel tubes. 
     A further aspect of the invention is to provide an automated transferring device for transferring samples from a container to an electrophoresis device where the transferring device includes a cover member having a plurality of apertures aligned in spaced apart rows and aligned with the electrophoresis device. 
     Another aspect of the invention is to provide an electrophoresis separation apparatus having a computer for controlling an electric power supply to the gel tanks and for the acquisition of run data for quality control. 
     The foregoing aspects and advantages of the invention are basically attained by providing an automated first dimensional electrophoresis separation apparatus comprising an electrophoresis assembly supporting a plurality of gel tubes containing an electrophoretic gel. Each of the tubes has a first open end and second open end and a supply magazine for containing a plurality of sample containers. Each sample container contains a sample to be subjected to electrophoresis. A transferring device is provided for sequentially removing a sample from a preselected sample container and transferring the sample to a first end of a respective gel tube. The transferring device includes a pipette that is movable in three dimensions between the supply magazine and a gel tube of the electrophoresis assembly. A microprocessor is operatively connected to the transferring device to automatically control the transfer of the sample to the respective gel tubes. 
     The aspects of the invention are further attained by providing an automated first dimension electrophoresis separation assembly comprising an electrophoresis assembly including at least one tank and a plurality of gel tubes vertically supported in the tank and arranged in a row. The gel tubes have an open top end. A supply magazine is provided for containing a plurality of sample containers. Each of the sample containers contains a liquid sample. A movable arm is movable in a substantially linear horizontal first direction between the supply magazine and the electrophoresis assembly. A movable pipette is coupled to the arm and is movable along a longitudinal dimension of the movable arm in a horizontal second direction substantially perpendicular to the first direction. The pipette is further movable in a vertical direction with respect to the movable arm. The pipette is movable from a first position for removing a sample from a sample container to a second position for dispensing a sample in a respective gel tube. 
     The aspects of the invention are still further attained by providing an apparatus for loading a biological sample in to an electrophoresis device. The apparatus comprises a base, a vertical support, and a stationary cover member spaced from the base. The cover has a top surface, a bottom surface and a plurality of apertures extending between the top and bottom surfaces and arranged in a plurality of spaced apart rows. The bottom surface of the cover member is positioned to receive a plurality of electrophoresis devices. The apparatus also includes a supply magazine for containing a plurality of sample containers that contain a biological sample. A robotic arm is movable between the supply magazine and a selected aperture of the cover member. The robotic arm has a pipette for withdrawing a sample from a sample container and delivering the sample through the aperture in the cover member to the electrophoresis device below the cover member. A microprocessor is operatively connected to the robotic arm for operating the robotic arm and the pipette. 
     The aspects, advantages and salient features of the invention will become apparent to one skilled in the art in view of the following detailed description of the invention in conjunction with the annexed drawings which form a part of this original disclosure. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The following is a brief description of the drawings, in which: 
     FIG. 1 is a perspective view of the apparatus of the invention showing the electrophoresis gel tanks, sample supply magazine and transferring device for transferring a sample from a sample container to a selected gel tube in a gel tank; 
     FIG. 2 is a top view of the apparatus of FIG. 1; 
     FIG. 3 is a perspective view of the supply magazine showing the carousel of the sample supply magazine, bar code reader and sample container holding device; 
     FIG. 4 is a partial top view of the apparatus of FIG. 1 showing the carousel and container retaining arm in a first position; 
     FIG. 5 is a partial top view of the apparatus of FIG. 1 showing the carousel and retaining arm in a second position for retaining a sample container in a holder; 
     FIG. 6 is a partial side view of the apparatus of FIG. 1 showing the movable arm and the actuating member for actuating the sample container holding device; 
     FIG. 7 is a partial side view of the sample container holding device showing the retaining arm positioned over the container; 
     FIG. 8 is a partial front view of the sample container holding device showing the retaining arm holding the sample container in place while the pipette penetrates the septum of the sample container; 
     FIG. 9 is perspective view of the electrophoresis tank and gel tube rack; 
     FIG. 10 is a front cross sectional view of the gel tube rack positioned in the tank in one embodiment of the invention; 
     FIG. 11 is a partial enlarged view in cross section of the rack showing the gasket for holding the gel tube in place; 
     FIG. 12 is an end view of the apparatus of FIG. 1 showing the electrophoresis tank and the apertures in the cover member for guiding the pipette into the gel tubes in the electrophoresis tank and showing the pipette in the raised position; 
     FIG. 13 is a partial cross sectional view of the apparatus of FIG. 8 with the electrophoresis tank positioned below the cover member and showing the pipette in the raised position above a gel tube; 
     FIG. 14 is a partial cross-sectional view of the tank and gel tube rack showing the pipette in the lowered position for transferring a sample into a gel tube; and 
     FIG. 15 is a schematic diagram of the assembly control system. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is directed to a method and apparatus for performing first dimension electrophoresis separation of a biological sample. In particular, the invention is directed to an automated apparatus for loading a plurality of samples into a respective tube containing an isoelectric gel and simultaneously performing electrophoresis separation of the samples. 
     The method and apparatus of the invention are used primarily in sequence with a second dimension electrophoresis separation step for isolating and recovering specific proteins in a sample. As discussed hereinafter in greater detail, the first dimension separation utilizes an electrophoresis gel in a tube having each end placed in contact with a buffer solution. An electric potential is applied across the ends of the gel tube to cause the proteins to migrate through the gel. The electrophoresis gel, such as IPG gels, and the buffer solutions are standard materials as known in the art of electrophoresis. 
     The biological samples to be subjected to the electrophoresis separation are typically protein samples. The protein samples are usually solubilized in an aqueous, denaturing solution such as 9 M urea, 2% NP-40 (a non-ionic detergent), 2% of a pH 8-10.5 ampholyte mixture and 1% dithiothreitol (DTT). The urea and NP-40 dissociate complexes of proteins with other proteins and with DNA and RNA. The ampholyte mixture establishes a high pH outside the range where most proteolytic enzymes are active and prevent modification of the sample protein by the ampholyte. The ampholyte further complexes with DNA present in the nuclei of sample cells and allows DNA-binding proteins to be released while preventing the DNA from swelling into a viscous gel that interferes with IEF separation. The dithiothreitol reduces the disulfide bonds in the proteins and allows them to unfold and assume an open structure that is more amenable for separation. Tissue samples are often solubilized by homogenizing in a solubilizing solution. The resulting mixture is centrifuged to remove insoluble material. 
     The method and apparatus of the invention are used in the first dimension separation of a two-dimensional separation system. The first dimension separation uses an isoelectric focusing gel, such as an acrylamide gel with a catalyst, focusing compounds and cross-linking agents. The gel is placed in a tube, such as a glass tube, having open ends. The bottom end of the tube is placed in a H 3 PO 4  buffer solution and the top end placed in a sodium hydroxide buffer solution to establish a pH gradient along the gel. The sample material is applied to the top end of the tube and allowed to migrate through the gel under the influence of an electrical potential. Generally, an electric current of about 1200 volts is applied between the upper and lower buffer solutions for about 20 hours. The isoelectric focusing gel and buffer solutions are conventional materials known in the art for first dimension separation. 
     Referring to the drawings, the electrophoresis apparatus  10  includes a sample supply magazine  12 , an automated robotic transferring assembly  14  and a plurality of electrophoresis tanks  16 . Tanks  16  contain several electrophoresis gel tubes that contain an isoelectric focusing gel. As discussed hereinafter, transferring assembly  14  automatically removes a biological sample from supply magazine  12  and robotically transfers and delivers the sample to a respective gel tube within tanks  16 . 
     Referring to FIGS. 3 and 4, supply magazine  12  in a preferred embodiment is mounted on a table  13  and includes a carousel  18  having a plurality of wells  20  for storing a plurality of sample containers  22 . Each sample container  22  is preferably a glass or plastic vial having an internal volume sufficient to contain a biological sample. A closure  24  is coupled to the open end  26  of sample container  22  to seal container  22  and prevent contamination of the sample and to prevent the sample from escaping. In a preferred embodiment, closure  24  is a flexible septum that can be pierced by a needle or pipette for withdrawing a sample from sample container  22 . 
     Carousel  18  includes a robotic arm  28  that is able to pivot around the center axis of carousel  18 . Carousel  18  is also able to rotate about its axis to bring a selected sample container into position for being picked up by robotic arm  28 . Robotic arm  28  is able to reciprocate in a radial direction with respect to carousel  18 . Robotic arm  28  includes a gripping member  30  that reciprocates in an up and down direction for gripping and removing a sample container  22  from a well  20  of carousel  18 . An example of this type of carousel is manufactured by the Hewlett-Packard Corporation as the HP Automatic Liquid Sampler, Model HP 18596B. 
     In one embodiment of the invention, supply magazine  12  includes a bar code reader  32  positioned adjacent carousel  18  for electronically reading, storing and indexing sample information. A suitable bar code reader is made by the Hewlett-Packard Corporation, such as the reader sold as model HPG 1926A. In alternative embodiments, other devices can be used for recording and storing information relating to the samples. Bar code reader  32  includes a well  34  for receiving a sample container  22 . Sample container  22  preferable includes a label  36  having a bar code or other indicia that can be read by bar code reader  32 . 
     Referring to FIG. 1 supply magazine  12  is connected to a central processing control unit  38  (CPU) such as a computer or microprocessor for controlling the movement of robotic arm  28  and recording information from bar code reader  32 . Central processing unit  38  actuates robotic arm  28  and carousel  18  to select a predetermined sample container  22  and remove sample container  22  from well  20  and transfer the container to bar code reader  32 . Bar code reader  32  records the information on label  36  and stores the information for tracking and identifying a sample throughout the separation process. Bar code reader  32  is operatively connected to central processing unit  38  for recording and tracking samples as depicted schematically in FIG.  15 . Supply magazine  12  also includes a sample container holding device  40  having a well  42  for receiving a sample container  22  from arm  28 . 
     As shown in FIGS. 3-5, holding device  40  is positioned adjacent carousel  18 . Arm  28  of supply magazine  12  is able to extend to a suitable length for retrieving a sample container  22  from carousel  18  and placing the sample container  22  into well  42 . Holding device  40  preferably includes a suitable mechanism for retaining sample container  22  in well  42  while the biological sample is removed from container  22 . 
     In a preferred embodiment shown in FIGS. 3,  4  and  5 , the retaining mechanism is a pivoting retaining arm  44  to hold sample container  22  within well  42 . Retaining arm  44  is mounted adjacent supply magazine  12  by a pivot pin  46  to allow retaining arm  44  to pivot about the axis of pin  46  from a first position shown in FIG. 4 to a retaining position shown in FIG.  5 . In the embodiment illustrated a spring  47  biases arm  44  away from supply magazine  12 . Retaining arm  44  includes an operating end  48  to hold sample container  22  in well  42 . In the illustrated embodiment, end  48  has an end plate  50  coupled thereto. End plate  50  is attached to retaining arm  44  by a fastener  52 . Preferably, fastener  52  is a threaded screw or bolt that can be tightened to fix the position of end plate  50  with respect to retaining arm  44  and can be loosened to enable end plate  50  to pivot to enable adjustment of end plate  50  to a desired location. In this manner, end plate  50  can be adjusted on retaining arm  44  to provide proper alignment of end plate  50  with respect to holding device  40  and well  42 . 
     As shown in FIG. 4, end plate  50  has an outer edge  54  with a substantially U-shaped recess  56 . End plate  50  has a dimension sufficient to overlie the top end of a sample container  22  when received in well  42  while exposing a portion of closure  24  of sample container  22  through recess  56  for piercing closure  42  by a piercing member to remove a sample from container  22 . In alternative embodiments, the retaining mechanism can be a gripping device able to grip the side walls of container  22 , or a vacuum source for drawing a vacuum sufficient to hold sample container  22  within well  42 . In other embodiments, plate  50  can be fixed to arm  44  or integrally formed therewith. Arm  44  can also be operated by a motor or piston and cylinder assembly, such as a pneumatic piston. A switch can be actuated by transferring assembly  14  to actuate the operating motor or pneumatic cylinder. 
     Referring to FIGS. 1 and 2, automated transferring assembly  14  includes a base  200  and two upright supports  202  that extend upwardly from the opposite rear corners of base  200 . A support  204  extends between upright supports  202  and is coupled to a top end of each upright support  202 . In the embodiment illustrated, upright supports  202  are substantially vertical and perpendicular to base  200 . Support  204  is horizontal and substantially parallel to base  200 . Support  204  includes a top face  208  having a track  210  extending in a longitudinal direction with respect to a longitudinal dimension of support  204  and a longitudinal dimension of assembly  14 . Generally, track  210  extends substantially the entire length of horizontal support  204 . In alternative embodiments, track  210  can be formed in the side or bottom of support  204 . 
     An arm  212  is coupled to support  204  and extends outwardly therefrom toward the front edge of base  200 . Arm  212  includes a first end  214  coupled to a drive and carriage assembly  216  for riding in track  210  of support  204 . Drive assembly  216  includes a suitable electrical motor (not shown) for moving arm  212  in the longitudinal direction of track  214 . The motor is connected to a suitable electric power source and to central processing unit  38  for controlling and operating the movement of arm  212 . Drive assembly  216  can be, for example, a gear drive or chain drive assembly connected to the motor for moving arm  212  in track  210  at a controlled speed and for controlling the precise position of arm  212  in track  214  relative to support  204 . In an alternative embodiment, carriage  216  can be coupled to a continuous belt that extends between two pulleys or gears at opposite ends of support  208 . A dual directional drive motor can be connected to one of the pulleys to move carriage  216  along support  208 . 
     In preferred embodiments arm  212  extends from support  204  in a substantially perpendicular direction with respect to the longitudinal dimension of support  204 . In alternative embodiments, arm  212  can be at an angle less then 90 degrees with respect to the longitudinal dimension of support  204 . Preferably, arm  212  is substantially parallel to base  200  and is coplanar with support  204 . 
     In one embodiment, arm  212  includes a track  220  enclosed within arm  212  as shown in FIG.  6 . Track  220  extends substantially the entire length of arm  212  and is dimensioned to support a carriage  222  for movement along the length of arm  212  in the longitudinal direction. Carriage  222  includes a motor and drive assembly  224  as shown in FIG. 6 for moving carriage along track  220 . Motor assembly  224  is connected to central processing unit  38  for controlling the movement and position of carriage on track  220 . Motor and drive assembly  224  can be a gear, belt or chain drive assembly capable of moving carriage  222  along track  220 . In alternative embodiments, the track can be provided on an external surface of arm  212 . 
     In one embodiment, track  220  can have a plurality of teeth for engaging a drive gear on motor assembly  224 . In an alternative embodiment, carriage  222  can be coupled to a continuous belt extending between pulleys at opposite ends of arm  212 . A drive motor can be connected to one of the pulleys for moving carriage  222  along track  220 . 
     As shown in FIG. 6, a pipette assembly  226  is coupled to carriage  222  for movement along track  220 . Pipette assembly  226  includes a support rod  228  coupled to carriage  222  and is positioned in a substantially vertical direction. Support rod  228  has a longitudinal dimension with a top end  230  and a bottom end  232 . A pipette  234  having a control valve member  236  is coupled to bottom end  232  of support rod  228 . Support rod  228  extends through carriage  222  and is coupled to a drive motor  238  for raising and lowering support rod  228  in a vertical direction with respect to arm  212 . In one embodiment of the invention support rod  228  includes external teeth for engaging a gear on motor  238  for raising and lowering support rod  228  with respect to arm  212 . Drive motor  238  is also connected to central processing unit  38  for operating support rod  228  as discussed hereinafter in greater detail. As shown in FIGS. 2 and 6 arm  212  includes an upper and lower longitudinal slot  240  extending the length arm  212  for allowing carriage  222  and support rod  228  to move along track  220 . In alternative embodiments carriage  222  for pipette assembly  226  can be mounted on an external surface of arm  212 . 
     Control valve member  236  preferably is an electrically operated valve for opening and closing pipette  234  to withdraw or dispense a liquid sample. Control valve member  236  and pipette  234  are coupled to a suitable pump through a flexible tube  242  for selectively providing a vacuum source and a pressure source for selectively withdrawing a liquid sample from sample container and dispensing the sample to a gel tube. Control valve member  236  and the pump are also connected to the central processing unit for operating pipette assembly  226 . 
     In preferred embodiments, pipette  234  is a hollow needle-like device having an axial length to be inserted into a supply container for withdrawing a liquid sample and for being inserted into or onto the end of a gel tube for dispensing the sample onto the open end of the gel tube. Typically pipette  234  is made of stainless steel or other materials that do not interfere with the sample materials. Preferably pipette  234  has an internal volume sufficient to contain a volume of a sample for conducting the electrophoresis separation without drawing the sample into the tube  242 . Since the required volume of a biological sample is quite small, pipette  234  is able to relieve a suitable volume for electrophoresis separation. In preferred embodiments pipette  234  has a sharpened tip  244  capable of penetrating the septum of a sample container so that a sample can be removed from a sample container without opening the sample container. 
     Referring to FIGS. 1,  2  and  6 , a support member  246  is coupled to arm  212  and extends in a direction substantially parallel to the longitudinal dimension of support  204 . In the embodiment illustrated support member  246  is substantially parallel to base  200 . An actuator rod  248  is coupled to support member  246  and extends in a downward direction toward base  200 . As shown in FIG. 6 actuator rod  248  is aligned with holding device  44  for moving arm  48  into the retaining position for retaining a sample container in the well  42 . 
     As shown in FIG. 1, transferring assembly  14  is coupled to central processing unit  38  for controlling the movement of movable arm  212 , carriage  222  and for operating pipette assembly  226 . In operation, sample containers  22  containing a biological sample are provided in carousel  18 . A sample container  22  is selected and grasped by arm  28  of carousel  18  and placed in bar code reader  32  where the sample identification and other information is recorded and stored in central processing unit  38 . Arm  28  of carousel  18  then transfers sample container  22  from bar code reader  32  to holding device  40 . Retaining arm  44  of supply magazine  12  is positioned in the horizontal path of actuator arm  248 . As shown in FIG. 6, actuator rod  248  of arm  212  is moved into contact with retaining arm  44  to pivot retaining arm  44  into the retaining position. 
     As shown in FIGS. 3 and 4, retaining arm  44  includes a bearing  82 , such as a roller bearing, for contacting actuator arm  248 . Retaining arm  44  also includes a biasing member, such as a spring  47 , to bias retaining arm  44  outwardly from carousel  18  to the position shown in FIG.  4 . As arm  212  is moved toward supply magazine  12  actuator arm  248  contacts bearing  82  causing retaining arm  44  to pivot about pivot pin  46  so that the end plate  50  overlies the sample container  22  as shown in FIG. 5 with U-shaped recess  56  oriented over closure  24 . Pipette assembly  226  is lowered to a position where pipette  234  pierces closure  24  of sample container  22 . Pump  80  is actuated to withdraw a desired amount of a sample from container  22  into pipette  234 . Pipette assembly  226  is then raised to withdraw pipette  234  from sample container  22 . End plate  50  of retaining arm  44  overlies sample container  22  to hold sample container  22  in holding device  40  while pipette  234  is withdrawn. Retaining arm  44  prevents sample container  22  from being lifted upward when pipette  68  is raised to the upper position. 
     Arm  212  and pipette assembly  226  are then moved along horizontal track  210  to a selected position corresponding to a designated gel tube in an electrophoresis tank  16 . As arm  212  is moved away from supply magazine  12 , actuator arm  248  disengages retaining arm  44 , allowing arm  44  to pivot outward from carousel  18 . Pipette assembly  226  is then lowered to a position at the top end of the designated gel tube and pump  256  is actuated to dispense the sample from pipette  234  onto the top end of the gel tube. Pipette assembly  236  is then raised and arm  212  is moved along horizontal track  210  to a rinsing station  250  for rinsing sample residue from pipette  234 . 
     Rinsing station  250  includes a container  252  containing a rinsing liquid such as distilled water. Pipette assembly  226  is lowered to insert pipette  234  into container  252  where a sufficient amount of the rinsing liquid is drawn into pipette  236  to rinse the inner surfaces of pipette  236 . Pipette  236  is then raised and moved to a position above a discharge container  254  where the rinsing liquid is discharged. Generally, a single rinsing cycle is sufficient to clean the residue from pipette  234 . 
     Arm  212  and pipette  236  are then moved back to the position shown in FIG.  5  and the steps repeated to transfer another sample from a sample container to a designated gel tube. The sequence of steps is repeated until the desired samples from the sample containers are transferred to a designated gel tube. Control unit  38  controls the movement of the supply magazine and transferring assembly  14  and records the location of each sample to identify a sample with a particular gel tube. 
     Assembly  10  includes a planar cover member  260  that is coupled to supports  202  at a rear edge thereof. Side supports  262  extend from the longitudinal ends  264  of cover member  260  to support the front and sides of cover member  260 . As shown in FIGS. 1 and 2 cover member  260  is substantially parallel to base  200 . Cover member  260  is dimensioned to overlie each electrophoresis tank  16  and is spaced from base  200  a distance to effectively close a top end of each electrophoresis tank  16 . As shown in FIG. 1 each electrophoresis tank  16  fits below cover member  260 . 
     Cover member  260  includes a plurality of apertures  266  oriented in parallel rows  268 . In a preferred embodiment of the invention rows  268  extend in a direction substantially perpendicular to the longitudinal dimension of support  204  and parallel to the longitudinal dimension of arm  212 . The number of apertures  266  in each row  268  correspond to the number of gel tubes in each electrophoresis tank  16  and are spaced apart to distance corresponding to the spacing between the gel tubes. As shown in FIG. 12 apertures  266  extend through cover member  260  and have inclined surfaces that converge to a bottom surface  272  of cover member  260 . In the embodiment illustrated, the inclined surfaces form a substantially frustoconical shaped top surface  270 . Frustoconical surfaces  270  are dimensioned to guide pipette  234  through apertures  266 . 
     A plurality of guide rails  274  are coupled to bottom surface  272  of cover member  260  as shown in FIGS. 1 and 2. Guide rails  274  extend in a direction substantially parallel to rows  268  of apertures  266 . In preferred embodiments of the invention guide rails  274  are oriented and spaced apart a distance to accurately position each electrophoresis tank  16  below cover member  260  so that each gel tube is positioned directly below a respective aperture  266 . An end wall  276  extends between adjacent guide rails  274  at a rear end of cover member  260  as shown in FIG.  12 . Guide rails  274  and end wall  276  serve as a guide assembly to position electrophoresis tank  16  for aligning the gel tubes with a respective aperture  266 . 
     Referring to FIGS. 9-14, electrophoresis tanks  16  have a bottom wall  280  and side walls  282  for containing a first buffer solution  283 . A rack  284  supporting a plurality of gel tubes  286  is dimensioned to fit within each tank  16  as shown in FIG.  10 . In one embodiment of the invention, bottom wall  280  of tank  16  can include an optional spacing member such as a pair of blocks for positioning rack  284  within tank  16  in a predetermined location. Preferably, tank  16  and rack  284  are dimensioned to fit between guide rails  274  and below cover member  260  with only minimal clearance. In this manner, rack  284  and gel tubes  286  are oriented in a precise location with respect to cover member  260  so that pipette  234  of transferring device  14  can transfer a biological sample from a sample container  22  to a designated gel tube  286  in successive runs without the need to recalibrate the apparatus after each run. In a preferred embodiment, gel tubes  286  are oriented in a straight row and spaced apart a distance corresponding to the spacing of apertures  266  in cover member  260 . 
     Rack  284  in the embodiment illustrated, has a pair of side walls  288  spaced apart a sufficient distance to enable rack  286  to fit within tank  16 . Side walls  288  function as a support for rack  286  when positioned in tank  16 . A lower brace  290  extends between side walls  288  to stabilize rack  284 . A plurality of spaced apart holes  292  having a conical surface are formed in brace  290  to support tubes  286  as shown in FIG.  10 . Preferably, brace  290  is a planar member extending perpendicular to side walls  288  to lie in a substantially horizontal plane when rack  284  is positioned in tank  16 . Brace  290  is coupled to side walls  288  by screws  296  or other suitable fasteners. A vertical brace  298  extends between side walls  288  and is coupled thereto by screws  300  or other suitable fasteners to further stabilize rack  284  as shown in FIGS. 10 and 13. 
     Rack  284  includes a top member  302  coupled to a top end  304  of side walls  282 . Top member  302  includes a lower plate  306  coupled together by screws  308 . Top member  302  includes a well  310  that is dimensioned to contain a sufficient amount of a second buffer solution  312  for conducting electrophoresis separation as known in the art. Well  310  is formed by a bottom wall  314  and side walls  316 . A ledge  318  extends outwardly from said walls  316  and is dimensioned to overlie the top end of side walls  282  of tank  16 . Lower plate  306  is oriented in a substantially horizontal position and parallel to bottom wall  314 . As shown in FIGS. 10 and 11, lower plate  306  is provided with a plurality of spaced apart openings  320  that are dimensioned to receive gel tubes  286 . Openings  320  have a conical recess  322  on a bottom face  324  of plate  306  for guiding gel tubes  286  into openings  320 . Plate  306  also includes an annular recess  326  on a top face  328  surrounding each opening  320  for receiving an annular gasket  330  having a substantially V-shaped cross-section. 
     Bottom wall  314  of well  310  includes a plurality of openings  332  having a conical shaped inlet end  334 . An annular recess  336  is formed in a bottom face  338  of bottom wall  314 . Annular recess  336  is dimensioned to receive the end of gel tube  286  as shown in FIG.  11 . 
     As shown in FIGS. 9 and 10, a first electrode  340  is provided within well  310  and secured in place by screws  342 . In a preferred embodiment of the invention, first electrode  340  is a wire that extends substantially the length of well  310 . As shown in FIG. 10, a second electrode  344  extends along brace  290  and is secured in place by mounting screws  346 . Electrode  344  is coupled to rack  284  in a position to be immersed in buffer solution  283 . 
     As shown in FIG. 10, ledge  318  of top member  302  is spaced from the bottom end of side walls  288  a distance corresponding substantially to the height of side walls  282  of tank  16 . In this manner, ledge  318  is able to rest on an upper end of side wall  282  with side walls  288  of rack  284  supported by bottom wall  280  of tank  16 . In one embodiment, alignment pins are provided in ledge  318  that are received in a respective recess formed in the top end of side wall  282  to orient rack  284  within tank  16 . In a preferred embodiment, the pins are spring loaded pins commonly referred to as “banana clips”. 
     In a preferred embodiment, two electrical contacts  348  in the form of pins extend outwardly from an end  350  of top member  302  as shown in FIGS. 9 and 10. Contact pins  348  are made of metal or other electrically conducting material. Electrodes  340  and  344  are connected to a respective contact pin  348 . End wall  276  at the end of guide rails  274  include two complementary contacts  352  having recesses for receiving contact pins  348 . Contacts  352  are connected to a suitable electric power source to apply an electric potential to electrodes  340  and  344 . Rack  284  is positioned between guide rails  274  and end  350  of ledge  318  rests against end wall  276  to enable contact pins  348  to engage contacts  352 . 
     Referring to FIG. 11, gel tubes  286  have a cylindrical shape with a central passage  354  and open ends  356 . The inner dimension of gel tubes  286  can range from 0.5 mm to about 2 mm and can be about 20 cm long. Gel tubes  286  are standard gel tubes as known in the electrophoresis art. An electrophoresis gel  358  is placed in gel tubes  286  to substantially fill the internal dimension as shown in FIG. 11 by known techniques. The gel forming materials can be placed in the tube and polymerized to form the gel. The gels can be IPG gels or other isoelectric focusing gels as known in the art. 
     The electrophoresis separation process of the invention is carried out using the apparatus  10 . Gel tubes  286  containing a gel  358  are mounted in rack  284  by sliding gel tubes  286  through the holes  292  in lower brace  290 . A conical surface of the holes  292  in lower brace  290  provide a guiding surface for guiding gel tubes  286  through brace  290 . Gel tubes  286  are then inserted into openings  320  of lower plate  306  using conical recesses  322  as a guide. The top end of gel tube  286  is seated in recess  336  of the bottom face of bottom wall  314  as shown in FIG.  11 . Annular gasket  330  is dimensioned to provide a fluid tight seal around gel tube  286  to prevent fluids from passing from well  310  into tank  16 . 
     A buffer solution  283 , such as a phosphoric acid solution, is provided in tank  16 . Rack  286  is positioned in tank  16  with a buffer solution  283  maintained at a level above the lower end of gel tubes  286  and electrode  340 . A second buffer solution  312 , such as a sodium hydroxide solution, is placed in well  310  to a sufficient level to cover the top end of gel tubes  286  and electrode  340 . Tank  16  is positioned between guide rails  274  to position each gel tube  286  directly below an aperture  266  in cover member  260 . 
     Transferring assembly  14  is actuated to transfer a biological sample from supply magazine  12  to a respective gel tube  286 . Pipette  234  withdraws a biological sample from a sample container  22  as previously discussed. Arm  212  moves along track  210  to a location above a respective gel tube  286  as shown in FIG.  13 . The conical surface  270  of aperture  266  guides pipette  234  through aperture  266  and directly to the top end of gel tube  286 . The conical surface  270  of apertures  266  forms a guide surface to compensate for misalignment of pipette  234  with aperture  266 . Although microprocessor  38  and the consistent location of gel tubes  286  usually provide proper alignment of pipette  234 , misalignment can occur as a result of the pipette tip being bent or distorted. Repeated piercing of the septum of the sample containers can bend pipette  234 , thereby causing the tip to be misaligned with the apertures  266  in cover  260 . Conical surfaces  270  can assist in aligning and directing the tip of pipette  234  to the proper location above gel tubes  286 . 
     As shown in FIG. 13, apertures  266  of cover  260  are axially aligned with openings  320  and gel tube  286 . As shown in FIG. 14, pipette assembly  226  is moved downward to insert the lower end of pipette  234  to the top end of gel tube  286 . Pipette  234  then dispenses the biological sample onto the top end of the gel in gel tube  286 . Pipette  234  is removed and returned to supply magazine  112  to repeat the process. 
     After a biological sample is placed on the top end of each gel tube  286 , contacts  252  are connected to a suitable power source  360  for applying an electric current to the electrodes and the buffer solutions. The electric current causes the various molecules of the biological sample to migrate through the gel tube as in standard first dimension electrophoresis separation. After a predetermined period of time, gel tubes  286  are removed from rack  284  and the gels are transferred to a second dimension separation apparatus as known in the art. 
     In preferred embodiments, power source  260  is operatively connected to central processing unit  38 . Central processing unit  38  controls the voltage applied between the electrodes  340 ,  344  of tank  16 . The current and voltage fluctuations are measured, continuously monitored and recorded over time throughout the duration of the isoelectric focusing to provide information for quality control. The recorded voltage and current can then be plotted as a function of time throughout the process. 
     FIG. 15 is a schematic diagram of the control system for coordinating the various operations discussed above. As shown, a central processing unit or computer indicated by block  364  is operatively connected to the carousel indicated by block  366  and bar code reader indicated by block  368  for recording data relating to each sample being processed. The movable arm motor indicated by block  370 , pipette carriage motor indicated by block  372  and pipette motor indicated by block  374  are connected to an controlled by the central processing unit. A pump indicated by block  376  is operatively connected to the central processing unit to control the operation of the pipette. A power source indicated by block  378  is also connected to the control processing unit to control the electrophoresis separation process. 
     A temperature control device is preferably provided with the tanks for measuring and adjusting the temperature of buffer solutions. The temperature control device is able to provide heating or cooling to the tank to maintain the temperature within a predetermined range. Preferably, temperature control device is connected to and controlled by central processing unit  38  through a suitable connection. 
     While various embodiments of the invention have been illustrated, it will be understood by those skilled in the art that additions and modifications can be made without departing from the scope of the invention as set forth in the appended claims.