Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2001-03-09
2004-07-13
Diamond, Alan (Department: 1753)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S456000, C204S465000, C204S606000, C204S615000, C204S610000, C422S063000, C422S064000, C422S065000, C422S067000, C436S043000
Reexamination Certificate
active
06761810
ABSTRACT:
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 ha
Anderson N. Leigh
Goodman Jack
McGrath Andrew
Diamond Alan
Large Scale Proteomics Corp.
Robbins John C.
Tarcza John E.
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