WO2002008741A1 - Dispositif automatise comprenant un bras robotise permettant de charger des echantillons dans des cupules pour separation par electrophorese de premiere dimension - Google Patents

Dispositif automatise comprenant un bras robotise permettant de charger des echantillons dans des cupules pour separation par electrophorese de premiere dimension Download PDF

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Publication number
WO2002008741A1
WO2002008741A1 PCT/US2001/022935 US0122935W WO0208741A1 WO 2002008741 A1 WO2002008741 A1 WO 2002008741A1 US 0122935 W US0122935 W US 0122935W WO 0208741 A1 WO0208741 A1 WO 0208741A1
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WO
WIPO (PCT)
Prior art keywords
sample
gel
rack
pipette
tank
Prior art date
Application number
PCT/US2001/022935
Other languages
English (en)
Inventor
N. Leigh Anderson
Andrew Mcgrath
Jack Goodman
Original Assignee
Large Scale Proteomics Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/621,484 external-priority patent/US6537434B1/en
Priority to AU2001276012A priority Critical patent/AU2001276012A1/en
Application filed by Large Scale Proteomics Corporation filed Critical Large Scale Proteomics Corporation
Publication of WO2002008741A1 publication Critical patent/WO2002008741A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44795—Isoelectric focusing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44743—Introducing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44782—Apparatus specially adapted therefor of a plurality of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices

Definitions

  • 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 test samples containing macromolecules from a sample well to a gel tube and conducting the first dimension isoelectric focusing of the sample.
  • IEF isoelectric focusing
  • Isoelectric separation is a known process that has been used for many years to separate macromolecules.
  • 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 that is in 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.
  • an electric current is applied to the ends of the tube, 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.
  • 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.
  • 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.
  • 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.
  • 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 samples containing proteins and other macromolecules.
  • 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 robotic arm where the arm is equipped with 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 robotic 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 from a sample container to a respective gel tube in the apparatus where the apparatus has a computer for recording identifying information and tracking the location of the samples through the apparatus.
  • 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 movable arm coupled to a support member. The movable arm is movable along a longitudinal dimension of the support member. A pipette is mounted on the movable arm and 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 for withdrawing a sample from a sample container to a sample-dispensing position for dispensing a sample to a gel tube.
  • a further aspect of the invention is to provide a computer automated apparatus for electrophoresis separation of macromolecules, where the apparatus has a plurality of electrophoresis gel tanks, each supporting a plurality 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, where the rack has an open well containing a buffer solution in contact with an end of the gel tube for electrophoresis separation.
  • the rack includes a guide for guiding a pipette to the 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 at least one sample container to a respective gel tube
  • the device includes a cover member positioned above an electrophoresis tank.
  • the cover member includes a plurality of apertures aligned with a respective number of gel tubes for supplying the samples to the gel tubes.
  • the cover is coupled to the electrophoresis tank.
  • the cover is fixed to a support and the electrophoresis tank is fitted below the support.
  • 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.
  • an automated first dimensional electrophoresis separation apparatus comprising an electrophoresis assembly including a tank and a plurality of gel tubes containing an electrophoretic gel supported in the tank.
  • Each of the tubes have a first open end in contact with a first buffer solution and a second open end in contact with a second buffer solution in the tank.
  • An electrical power source has a first electrode in the first buffer solution and a second electrode in the second buffer solution.
  • a supply magazine for containing a plurality of sample containers is provided. Each sample container contains a sample to be subjected to electrophoresis separation.
  • An automated transferring device sequentially removes a sample from a preselected sample container and transfers the sample to a first end of a respective gel tube.
  • a microprocessor is connected to the transferring device to automatically control the transfer of the samples to the respective gel tubes.
  • an automated first dimension electrophoresis separation assembly comprising a rack which has an upper end, a lower end, and a chamber formed in the upper end for containing a first buffer solution.
  • the chamber has a bottom wall with a plurality of spaced-apart openings.
  • the rack includes at least two spaced-apart pins.
  • a plurality of gel tubes is provided. Each of the tubes has a first end received in a respective opening in the bottom wall of the chamber.
  • a plurality of electrophoresis gel tubes is coupled to the rack. Each of the gel tubes has a first end received in a respective opening in the bottom wall of the chamber.
  • a tank for containing a second buffer solution is provided, and the tank has at least one side wall with a top end which has at least two spaced-apart apertures therein.
  • the apertures are dimensioned to complement the spaced apart pins of the rack for orienting the rack in a predetermined location in the tank.
  • the aspects of the invention are still further attained by providing an apparatus for supporting a plurality of gel tubes in an electrophoresis assembly.
  • the rack comprises at least one support member for supporting the rack in an electrophoresis tank, and a bottom wall having a plurality of spaced-apart openings.
  • the openings are dimensioned to receive an end of an electrophoresis gel tube.
  • At least one side wall is coupled to the bottom wall to form a chamber dimensioned for containing an electrophoresis buffer solution.
  • the chamber is in communication with the end of each of the electrophoresis gel tubes.
  • the aspects of the invention are yet further attained by providing a computer controlled method for the automated first dimensional electrophoresis separation of a sample containing macromolecules.
  • the method comprises the steps of: providing a plurality of the samples in sample containers, robotically removing the samples from the sample containers by a computer controlled robotic assembly and delivering the samples to one end of a respective isoelectric focusing gel, and applying an electric current to the isoelectric focusing gel for providing an electric potential between opposite ends of the gel and separating macromolecules of the samples within the isoelectric focusing gel.
  • an automated first dimensional electrophoresis separation apparatus comprising an electrophoresis assembly including a tank, a rack positionable in the tank, a plurality of gel tubes containing an isoelectric focusing gel supported by the rack, and a supply magazine for containing a plurality of sample containers.
  • Each sample container contains a sample to be subjected to electrophoresis separation.
  • a transferring device for sequentially removing a sample from a preselected sample container and transferring the sample to a first end of a respective gel tube is provided.
  • a container holding member for holding a pre-selected sample container while the transferring device removes a sample from the sample container is also provided.
  • a microprocessor controls the transferring device and automatically controls the transfer of the samples to the respective gel tubes.
  • Figure 1 is a perspective view of the apparatus of the invention in a first embodiment showing the gel tanks, sample supply magazine and transferring device for transferring a sample from a sample container to a gel tube in a gel tank;
  • Figure 2A is a front view of the apparatus of Figure 1 in partial cross-section;
  • Figure 2B is a top view of the apparatus of Figure 1;
  • Figure 3 is a perspective view of the supply magazine showing the carousel, bar code reader and sample container holding device;
  • Figure 4 is a partial top view of the apparatus of Figure 1 showing the carousel and container retaining arm in a first position;
  • Figure 5 is a partial top view of the apparatus of Figure 1 showing the carousel and retaining arm in a second position for retaining a sample container in a holder;
  • Figure 7 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;
  • Figure 8 is a front elevational view of the gel tube rack positioned in the tank in a preferred embodiment of the invention.
  • Figure 9 is an enlarged area in cross section of the rack showing the gasket for holding the gel tube in place;
  • Figure 10 is a top view of the gel tube rack of Figure 8 with the top cover removed and showing the chamber for a buffer solution;
  • Figure 11 is a side view of the rack of Figure 8.
  • Figure 12 is a partial cross-sectional view of the tank and gel tube rack showing the pipette positioned above the rack;
  • Figure 13 is a partial cross-sectional view of the tank and gel tube rack showing the pipette inserted through the openings in the rack for delivering a sample to the gel tube;
  • Figure 14 is a perspective view of the apparatus of the invention in a second embodiment 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;
  • Figure 15 is a top view of the apparatus of Figure 14;
  • Figure 16 is a partial top view of the apparatus of Figure 14 showing the carousel and container retaining arm in a first position;
  • Figure 1 is a partial top view of the apparatus of Figure 14 showing the carousel and retaining arm in a second position for retaining a sample container in a holder;
  • Figure 18 is a partial side view of the apparatus of Figure 14 showing the movable arm and the actuating member for actuating the sample container holding device;
  • Figure 19 is perspective view of the electrophoresis tank and gel tube rack
  • Figure 20 is a front cross sectional view of the gel tube rack positioned in the tank in one embodiment of the invention.
  • Figure 21 is a partial enlarged view in cross section of the rack showing the gasket for holding the gel tube in place;
  • Figure 22 is an end view of the apparatus of Figure 14 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;
  • Figure 23 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;
  • Figure 24 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;
  • Figure 25 is a schematic diagram of the assembly control system.
  • the present invention is directed to a method and apparatus for performing first dimension electrophoresis separation of a biological sample.
  • 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 sample.
  • 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.
  • the first dimension separation utilizes an electrophoresis gel in a tube having each end placed in a buffer solution. An electric potential is applied to cause the proteins to migrate through the gel.
  • the electrophoresis gel and the buffer solutions are standard materials, such as IPG gels, 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 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 H3PO4 buffer solution and the top end is 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.
  • the electrophoresis apparatus 10 includes a sample supply magazine 12, an automated robotic transferring assembly 14 and a plurality of electrophoresis buffer tanks 16.
  • Buffer tanks 16 contain several tubes that contain an isoelectric focusing gel.
  • transferring assembly 14 automatically removes a sample from supply magazine 12 and robotically transfers and delivers the sample to the tubes within tanks 16.
  • supply magazine 12 in a preferred embodiment is 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.
  • closure 24 is a flexible septum that can be pierced by a needle or pipette to withdraw a sample from sample container 22.
  • Carousel 18 includes a robotic arm 28 that is able to pivot around the axis of carousel 18. Carousel 18 is also able to rotate about its axis to bring a selected sample container into position to be picked up by robotic arm 28. Robotic arm 28 is also 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.
  • 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.
  • Supply magazine 12 is connected to a central processing control unit 38 (CPU) such as a microprocessor for controlling the movement of robotic arm 28 and recording information from bar code reader 32.
  • CPU central processing control unit 38
  • 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.
  • Supply magazine 12 also includes a sample container holding device 40 having a well 42 for receiving a sample container 22 from arm 28.
  • holding device 40 is positioned adjacent carousel 18. Arm 28 is able to reciprocate a suitable length for placing a 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.
  • the retaining mechanism is a retaining arm 44 provided on supply magazine 12 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 Figure 4 to a retaining position shown in Figure 5.
  • 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.
  • end 48 has an end plate 50 coupled thereto. End plate 50 is attached to retaining arm 44 by a fastener 52.
  • 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.
  • 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.
  • 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.
  • 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.
  • automated transferring assembly 14 is positioned adjacent supply magazine 12 and includes a movable arm assembly 58.
  • Movable arm assembly 58 includes a main body 60 that is movable along a horizontal track 62.
  • Transferring assembly 14 includes a drive mechanism driven by a suitable motor for moving body 60 along track 62.
  • the drive mechanism can be, for example, a gear or chain drive assembly connected to the motor for moving main body 60 at a controlled speed and precisely controlling the position of main body 60 along track 62.
  • Body 60 is provided with a vertical track 64 in which an operating arm 66 is received and movable in a vertical direction.
  • Operating arm 66 supports a pipette 68 oriented vertically and coupled to arm 66 for reciprocating movement with arm 66.
  • Pipette 68 is a micropipette having a generally cylindrical shape with an axial passage extending therethrough.
  • a distal end 70 includes a sharpened tip 72 as shown in Figure 6.
  • a top end 74 includes a coupling 76 attached to a flexible tube 78.
  • Tube 78 is connected to a pump 80 shown in Figure 2A for selectively applying a negative pressure to pipette 68 for drawing a vacuum to collect a sample and for applying a positive pressure to dispense the sample from the pipette.
  • pipette 68 is a metal syringe needle-like device having an internal volume sufficient to contain a biological sample for a first dimension electrophoresis separation.
  • transferring assembly 14 is also coupled to central processing unit 38 for controlling the movement of movable arm assembly 58 and the actuation of pump 80.
  • sample containers containing a biological sample are provided in carousel 18.
  • Arm 28 of carousel 18 selects a sample container 22 from carousel 18 and places sample container 22 in bar code reader 32 where the sample identification and other information is recorded and stored in control unit 38.
  • Arm 28 of carousel 18 then transfers sample container 22 from bar code reader 32 to holding device 40.
  • main body 60 of transferring assembly 14 slides along horizontal track 62 from a position adjacent holding device 40 to a respective buffer tank 16 and a designated tube mounted in tank 16.
  • Retaining arm 44 of supply magazine 12 is positioned in the horizontal path of main body 60 as main body 60 moves along track 62 in the direction of supply magazine 12.
  • retaining arm 44 includes a bearing 82, such as a roller bearing, for contacting main body 60.
  • 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 Figure 4.
  • a biasing member such as a spring 47
  • main body 60 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 Figure 5 with U-shaped recess 56 oriented over closure 24.
  • Movable arm 58 is lowered to a position where pipette 68 pierces closure 24 of sample container 22.
  • Pump 80 is actuated to withdraw a desired amount of a sample from container 22 into pipette 68.
  • Movable arm 58 is then raised to withdraw pipette 68 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 68 is withdrawn.
  • Retaining arm 44 prevents sample container 22 from being lifted upward when pipette 68 is raised to the upper position.
  • Main body 60 is then moved along horizontal track 62 a selected position corresponding to a designated gel tube in a tank 16. As body 60 is moved away from supply magazine 12, body 60 disengages retaining arm 44, allowing arm 44 to pivot outward from carousel 18.
  • Movable arm 58 is then lowered to a position at the top end of the designated tube and pump 80 is actuated to dispense the sample from pipette 68 onto the top end of the gel tube. Movable arm 58 is then raised and moved along horizontal track to a rinsing station 84 shown in Figures 2A and 2B for rinsing sample residue from pipette 68.
  • Rinsing station 84 includes a container 86 containing a rinsing liquid such as distilled or de-ionized water. Movable arm 58 is lowered to insert pipette 68 into container 86 where a sufficient amount of the rinsing liquid is drawn into pipette 68 to rinse the inner surfaces of pipette 68. Pipette 68 is then raised and moved to a position above a discharge container 88 where the rinsing liquid is discharged.
  • a rinsing liquid such as distilled or de-ionized water.
  • Movable arm 58 and pipette 68 are then moved back to the position shown in Figure 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.
  • electrophoresis buffer tanks 16 have a bottom wall 100 and side walls 102 for containing a first buffer solution.
  • a rack 104 supporting a plurality of gel tubes 106 is dimensioned to fit within tank 16 as shown in Figure 6.
  • bottom wall 100 of tank 16 can include an optional spacing member such as a pair of blocks 108 for positioning rack 104 within tank 16 in a predetermined location.
  • tank 16 is fixed to a support surface or positioned in a specific location with respect to assembly 14.
  • rack 104 and gel tubes 106 are oriented in a precise location with respect to assembly 14 so that the arm of transferring device 28 can transfer a biological sample from a sample container 22 to a designated gel tube in successive runs without the need to recalibrate the apparatus after each run.
  • gel tubes 106 are oriented in a straight line aligned with the pipette 68 so that pipette 68 can be positioned above each tube 106 by selectively moving main body 60 of transferring device 14 along track 62. Pipette 68 can then be lowered along straight track 64 to dispense the sample to a gel tube.
  • Rack 104 in the embodiment illustrated has a pair of side walls 110 spaced apart a sufficient distance to enable rack 104 to fit within tank 16.
  • Side walls 110 function as a support for rack 104 when rack 104 is positioned in tanks 16.
  • a lower brace 112 extends between side walls 110 to stabilize rack 104.
  • a plurality of spaced apart holes 105 having a conical surface 107 are formed in brace 112 to support tubes 106 as shown in Figure 12.
  • brace 112 is a planar member extending perpendicular to side walls 110 to lie in a substantially horizontal plane when rack 104 is positioned in tank 16.
  • Brace 112 is coupled to side walls 110 by screws 114 or other suitable fasteners.
  • a vertical brace 116 extends between side walls 110 and is coupled thereto by screws 118 or other suitable fasteners to further stabilize rack 104.
  • Rack 104 includes a trough assembly 120 coupled to a top end 122 of side walls 110.
  • Trough assembly 120 includes a lower plate 124, a middle block 136 and a top plate 176.
  • Trough 120 includes a chamber that is dimensioned to contain a sufficient amount of a second buffer solution for conducting electrophoresis separation as known in the art.
  • Trough 120 includes a lower plate 124 that is attached to side walls 110.
  • Lower plate 124 is oriented in a substantially horizontal position and parallel to lower brace 112. As shown in Figures 8 and 9, lower plate 124 is provided with a plurality of spaced apart openings 126 that are dimensioned to receive gel tubes 106.
  • Openings 126 have a conical recess 128 on a bottom face 130 of plate 124 for guiding tubes 106 into openings 126.
  • Plate 124 also includes an annular recess 132 on a top face 131 surrounding each opening 126 for receiving an annular gasket 134 having a substantially N-shaped cross-section.
  • Middle block 136 of trough 120 is coupled to lower plate 124 by screws 138.
  • Block 136 has a bottom face 140 for mating with plate 124.
  • a top face 142 of block 136 includes a recessed area 144 defining an upper chamber or tank for containing the second buffer solution.
  • Recessed area 144 has a bottom face 146 and side walls 148.
  • bottom face 146 is substantially parallel to top face 142 and side walls 148 are perpendicular to bottom face 146 and top face 142.
  • Recessed area 144 includes a plurality of openings 150 extending from bottom face 146 of recessed area 144 to bottom face 140 of middle block 136. Openings 150 have a conical shaped inlet end 152 formed in face 146 and an annular recess 154 in bottom face 140. Annular recess 154 is dimensioned to receive the end of gel tube 106.
  • first electrode 156 is provided within recessed area 144 and secured in place by screws 158.
  • first electrode 156 is a wire that extends substantially the length of recessed area 144.
  • a second electrode 160 extends along brace 112 and is secured in place by mounting screws 162.
  • Block 136 includes a ledge portion 164 extending outwardly from a top end in a direction generally parallel to bottom face 140. As shown in Figures 11 and 13, ledge 164 is spaced from the bottom end of side walls 110 a distance corresponding substantially to the height of side walls 102 of tank 16. In this manner, ledge 164 is able to rest on an upper end of side wall 102 with side walls 110 of rack 104 supported by bottom wall 100 of tank 16. In a preferred embodiment, at least two alignment pins 166 extend downwardly through ledge 164. Each pin 166 has a lower portion 168 that is received in a respective recess 170 formed in the top end of side wall 102.
  • Recesses 170 in side wall 102 are located to orient rack 104 in a specific location within tank 16. Since tank 16 is mounted in a fixed position, rack 104 can be removed from tank 16 and replaced in the same orientation for transferring assembly 14 to consistently deliver a sample to a gel tube supported by rack 104.
  • Pin 166 can be a cylindrical shape or square. In a preferred embodiment, pins 166 are spring loaded pins commonly referred to as "banana clips”. [0078]
  • each recess 170 in the top end of side walls 102 include a metal sleeve 172 that is connected to a coupling member 174 as shown in Figure 13. Pins 166 are made of metal or other electrically conducting material for making electrical contact with sleeves 172.
  • Electrodes 156 and 160 are connected to a respective pin 166 so that rack 104 can be positioned in tank 16 to provide an electrical connection between coupling member 174 and electrodes 156 and 160.
  • Coupling members 174 are connected to a suitable electric power source to apply an electric potential to electrodes 156 and 160.
  • Trough assembly 120 includes a top wall 176 that is removably coupled to top face 142 of block 136 by pins 178 as shown in Figure 11.
  • Top wall 176 is provided with apertures 180 complementing alignment pins 166.
  • top wall 176 is coupled to block 136 and oriented by alignment pins 166.
  • Top wall 176 also includes a plurality of spaced apart apertures 190 aligned with the openings 150 in bottom face 146.
  • a top face of top wall 176 includes conical shaped recesses 192 axially aligned with apertures 190 to form guiding surfaces for directing the pipette into apertures 190.
  • gel tubes 106 have a cylindrical shape with a central passage 182 and open ends.
  • the inner dimension of gel tubes 106 can range from 0.5 mm to about 2 mm and can be about 20 cm long.
  • Gel tubes 106 are standard gel tubes as known in the art.
  • An electrophoresis gel 184 is placed in gel tubes 106 to substantially fill gel tubes 106 as shown in Figure 9 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 106 containing a gel 184 are mounted in rack 104 by sliding gel tubes 106 through the holes 105 in lower brace 112.
  • the conical surface 107 of the holes 105 in lower brace 112 provides a guiding surface for guiding gel tubes 106 through brace 112.
  • Gel tubes 106 are then inserted into openings 126 of lower plate 124 using conical recesses 128 as a guide.
  • the top end of gel tube 106 is seated in recess 132 of bottom face 130 as shown in Figure 11.
  • Annular gasket 134 is dimensioned to provide a fluid tight seal around gel tube 106 to prevent fluids from passing from trough 120 into tank 16.
  • a gel tube 106 is positioned in each respective opening in rack 104.
  • a buffer solution 186 such as a phosphoric acid solution, is provided in tank 16 and rack 104 is positioned in gel tank 16 with alignment pins 166 received in recesses 170 of tank 16. Buffer solution 186 is maintained at a level above the lower end of gel tubes 106 and electrode 160.
  • a second buffer solution 188 such as a sodium hydroxide solution, is placed in trough 120 to a sufficient level to cover the top end of gel tubes 106 and first electrode 156.
  • Top wall 176 is coupled to block 136 to close the chamber.
  • transferring assembly 14 is actuated to transfer a biological sample from supply magazine 12 to a respective gel tube 106.
  • Pipette 68 withdraws a biological sample from a sample container 22 as previously discussed.
  • Main body 60 of assembly 14 moves along track 62 to a location above a respective gel tube 106 as shown in Figure 9.
  • Top wall 176 of trough 120 is provided with a plurality of apertures 190 corresponding to each gel tube in rack 104.
  • Apertures 190 have a conical top end 192 for guiding pipette 186 through aperture 190.
  • the conical surface 192 of aperture 190 forms a guide surface to compensate for misalignment of pipette 168 with aperture 190.
  • microprocessor 38 and the consistent location of racks 104 and gel tubes 106 usually provide proper alignment of pipette 168, 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 168, thereby causing the tip to be misaligned with the openings in rack 104.
  • Conical surfaces 152 and 192 can assist in aligning and directing the tip of pipette 168 to the proper location above gel tubes 106.
  • apertures 190 of top wall 176 are axially aligned with openings 150 and gel tube 106.
  • Movable arm 58 of assembly 14 is moved downward to insert the lower end of pipette 68 to the top end of gel tube 106.
  • Conical surface 152 of opening 150 serves to guide pipette 68 to gel tube 106.
  • Pipette 68 then dispenses the biological sample onto the top end of gel 184 iri gel tube 106. Pipette 68 is removed from rack 104 and returned to supply magazine 112 to repeat the process.
  • coupling members 174 are connected to a suitable power source 194 for applying an electric current to the electrodes and the buffer solutions.
  • the electric current causes the various components of the biological sample to migrate through the gel tube as in standard first dimension electrophoresis separation.
  • gel tubes 106 are removed from rack 104 and the gels are transferred to a second dimension separation apparatus as known in the art.
  • power source 194 is operatively connected to central processing unit 38.
  • Central processing unit 38 controls the voltage applied to the electrodes 156, 160 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.
  • a temperature control device 196 is preferably provided with tanks 16 as shown in Figure 12 for measuring and adjusting the temperature of buffer solution 186 in tank 16. Temperature control device 196 is able to provide heating or cooling to tank 16 to maintain the temperature within a predetermined range. Preferably, temperature control device 196 is connected to and controlled by central processing unit 38 through a connection 198.
  • Figures 14-25 show a second embodiment of the invention for a computer controlled apparatus 200 for loading samples into electrophoresis gel tubes and conducting electrophoresis separation of the samples.
  • Apparatus 200 is similar to the embodiment of Figures 1-13 so that identical components are identified by the same reference number with the addition of a prime.
  • the electrophoresis apparatus 200 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.
  • 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'.
  • supply magazine 12' is mounted on a table 13' and 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 a reading device for reading a symbol or symbols on a sample container such as a bar code reader 32'.
  • Central processing control unit 38' continuously reads, records and stores data for identifying and indexing a sample and monitoring the location of the sample during the electrophoresis process.
  • central processing unit 38' actuates robotic arm 28' and carousel 18' to select a predetermined sample container, remove the sample container from a well, and transfer the container to well 34' of bar code reader 32'.
  • Supply magazine 12' also includes a sample container holding device 40' having a well 42' for receiving a sample container from arm 28'.
  • the device 40' includes a pivoting retaining arm 44' mounted adjacent supply magazine 12' by a pivot pin 46'.
  • a spring 47' biases arm 44' away from supply magazine 12'.
  • retaining arm 44' includes an operating end 48' having an end plate 50' coupled thereto by a fastener 52'.
  • End plate 50' has an outer edge 54' with a substantially U-shaped recess 56'.
  • automated transferring assembly 14' includes a base 201 and two upright supports 202 that extend upwardly from the opposite rear corners of base 201.
  • a support 204 extends between upright supports 202 and is coupled to a top end of each upright support 202.
  • upright supports 202 are substantially vertical and perpendicular to base 201.
  • Support 204 is horizontal and substantially parallel to base 201.
  • 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 200.
  • track 210 extends substantially the entire length of horizontal support 204.
  • 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 201.
  • 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.
  • 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.
  • 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 201 and is coplanar with support 204. [0095] In one embodiment, arm 212 includes a track 220 enclosed within arm 212 as shown in Figure 18. 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 Figure 18 for moving carriage along track 220.
  • Motor assembly 224 is connected to central processing unit 38' for controlling the movement and position of carriage 222 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.
  • the track can be provided on an external surface of arm 212.
  • track 220 can have a plurality of teeth for engaging a drive gear on motor assembly 224.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 256 through a flexible tube 242 for selectively providing a vacuum source and a pressure source for selectively withdrawing a liquid sample from a sample container and dispensing the sample to a gel tube.
  • Control valve member 236 and the pump 256 are also connected to the central processing unit 38' for operating pipette assembly 226.
  • 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.
  • pipette 234 is made of stainless steel or other materials that do not interfere with the sample materials.
  • 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 receive a suitable volume for electrophoresis separation.
  • 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.
  • a support member 246 is coupled to arm 212 and extends in a direction substantially parallel to the longitudinal dimension of support 204.
  • 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 201.
  • 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'.
  • 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.
  • 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' as in the previous embodiment.
  • Arm 28' of carousel 18' then transfers sample container 22' from bar code reader 32' to holding device 40' and actuator rod 248 of arm 212 is moved into contact with retaining arm 44' to pivot retaining arm 44' into the retaining position.
  • 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 Figure 16.
  • 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 Figure 17 with U-shaped recess 56' oriented over closure 24'.
  • Retaining arm 44' is the same as in the previous embodiment and overlies the sample container to hold the sample container while pipette 234 is withdrawn.
  • 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'.
  • 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 Figure 17 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 200 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 Figures 14 and 15, cover member 260 is substantially parallel to base 201. 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 Figure 14, each electrophoresis tank 16' fits below cover member 260.
  • Cover member 260 includes a plurality of apertures 266 oriented in parallel rows 268.
  • 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.
  • apertures 266 extend through cover member 260 and have inclined surfaces 270 that converge to a bottom surface 272 of cover member 260.
  • the inclined surfaces 270 form a substantially frustoconical shaped top surface. Inclined 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 Figures 14 and 15.
  • Guide rails 274 extend in a direction substantially parallel to rows 268 of apertures 266.
  • 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 Figure 22.
  • 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.
  • 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 Figure 20.
  • 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.
  • tank 16' and rack 284 are dimensioned to fit between guide rails 274 and below cover member 260 with only minimal clearance.
  • 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.
  • 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 Figure 20.
  • 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 Figures 20 and 23.
  • 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 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 Figures 20 and 21, 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 Figure 11.
  • first electrode 340 is provided within well 310 and secured in place by screws 342.
  • first electrode 340 is a wire that extends substantially the length of well 310.
  • 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.
  • 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.
  • 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'.
  • 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'.
  • the pins are spring loaded pins commonly referred to as "banana clips”.
  • two electrical contacts 348 in the form of pins extend outwardly from an end 350 of top member 302, as shown in Figures 19 and 20.
  • 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.
  • 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 Figure 21 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 200.
  • 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 Figure 21.
  • 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'.
  • 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.
  • 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 Figure 23.
  • 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.
  • control processing unit 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.
  • apertures 266 of cover 260 are axially aligned with openings 320 and gel tube 286.
  • 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 12' to repeat the process.
  • each gel tube 286 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.
  • 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. 25 is a schematic diagram of the control system for coordinating the various operations discussed above.
  • 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 and 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.
  • temperature control device is connected to and controlled by central processing unit 38' through a suitable connection.

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Abstract

L'invention concerne un montage automatise (10) permettant de réaliser une séparation par électrophorèse de première dimension de protéines ou d'autres macromolécules, comprenant un chargeur d'échantillons (12); un dispositif de transfert automatisé (14); et une cuve à électrophorèse (16). La cuve (16) comprend, d'une part, une grille sur laquelle reposent une pluralité de tubes de gel, et d'autre part, une chambre conçue pour contenir une solution tampon, en contact avec une extrémité des tubes de gel. Le montage comprend un élément couvercle recouvrant la cuve. Le chargeur d'alimentation comprend un carrousel permettant de stocker les contenants d'échantillon; un lecteur de codes à barres, un dispositif de maintien et un bras permettant de transférer le contenant d'échantillons depuis le carrousel vers le lecteur de codes à barres et le dispositif de maintien. Le dispositif de transfert comprend un ensemble robotisé (58) qui peut être déplacé en trois dimensions, de manière à retirer un échantillon d'un contenant d'échantillon et à distribuer ledit échantillon à un tube de gel sélectionné dans la cuve à électrophorèse.
PCT/US2001/022935 2000-07-21 2001-07-20 Dispositif automatise comprenant un bras robotise permettant de charger des echantillons dans des cupules pour separation par electrophorese de premiere dimension WO2002008741A1 (fr)

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US09/621,484 2000-07-21
US09/621,484 US6537434B1 (en) 2000-07-21 2000-07-21 First dimension electrophoresis separation method and apparatus
US09/801,831 2001-03-09
US09/801,831 US6761810B2 (en) 2000-07-21 2001-03-09 Automated apparatus including a robotic arm for loading samples into wells for first dimension electrophoresis separation

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10087436B2 (en) 2014-02-06 2018-10-02 The Regents Of The University Of California Electrophysiologically mature cardiomyocytes and methods for making same
CN113804909A (zh) * 2020-06-12 2021-12-17 中国科学院苏州纳米技术与纳米仿生研究所 真空互联样品转移组件

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4048049A (en) * 1976-11-19 1977-09-13 Hoefer Scientific Instruments Tube gel electrophoresis device
US4284491A (en) * 1978-11-13 1981-08-18 C. Desaga Gmbh Nachf. Erich Fecht Apparatus for electrophoresis
US5993627A (en) * 1997-06-24 1999-11-30 Large Scale Biology Corporation Automated system for two-dimensional electrophoresis

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4048049A (en) * 1976-11-19 1977-09-13 Hoefer Scientific Instruments Tube gel electrophoresis device
US4284491A (en) * 1978-11-13 1981-08-18 C. Desaga Gmbh Nachf. Erich Fecht Apparatus for electrophoresis
US5993627A (en) * 1997-06-24 1999-11-30 Large Scale Biology Corporation Automated system for two-dimensional electrophoresis

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10087436B2 (en) 2014-02-06 2018-10-02 The Regents Of The University Of California Electrophysiologically mature cardiomyocytes and methods for making same
CN113804909A (zh) * 2020-06-12 2021-12-17 中国科学院苏州纳米技术与纳米仿生研究所 真空互联样品转移组件
CN113804909B (zh) * 2020-06-12 2023-12-12 中国科学院苏州纳米技术与纳米仿生研究所 真空互联样品转移组件

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