Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2000-07-21
2003-03-25
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S456000, C204S465000, C204S606000, C204S610000, C204S615000, C422S063000, C422S064000, C422S067000, C436S043000
Reexamination Certificate
active
06537434
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to a method and apparatus for performing isoelectric focusing of macromolecules, and particularly proteins. More particularly, the present invention is directed to an automated apparatus for the first dimension isoelectric focusing of proteins.
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 acrylamide, is placed or polymerized in a tube and positioned in a bath with a buffer solution at each end. One buffer solution is typically a sodium hydroxide solution. The other buffer solution is typically a phosphoric acid solution. 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 a pH gradient 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 two-dimensional electrophoresis separations. 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 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. Generally, 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 dimensional isoelectric focusing.
SUMMARY OF THE INVENTION
The present invention is directed to an apparatus for the electrophoresis separation of macromolecules and particularly proteins. More particularly, the invention is directed to an apparatus for first dimensional isoelectric focusing of proteins and other macromolecules.
Accordingly, a primary object of the invention is to provide an automated apparatus for preparing samples for electrophoresis separation.
Another object of the invention is to provide an automated apparatus for sequentially transferring a biological sample from a sample container to a gel tube for performing electrophoresis separation of the sample.
A further object of the invention is to provide an automated apparatus for transferring a biological sample from a sample container to a gel tube where the sample container is identified and selected from a container supply magazine.
Another object 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 while a sample is being removed.
A further object 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 sample from the container.
Still another object 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 container while the pipette penetrates and is withdrawn from a septum in a sample container.
Another object of the invention is to provide an automated apparatus for transferring a plurality of sample solutions to a respective gel tube and recording and tracking the location of the samples.
A further object of the invention is to provide an automated apparatus for transferring a plurality of sample solutions to a respective gel tube, wherein the apparatus includes a pipette mounted on an arm that is movable vertically for withdrawing a sample from a container and for dispensing a sample to a gel tube.
Another object of the invention is to provide an automated apparatus for electrophoresis separation having a movable robotic arm and a pipette that is movable from a sample withdrawing position to a sample dispensing position.
A further object of the invention is to provide an automated apparatus for electrophoresis separation having a movable robotic arm, where movement of the arm actuates a holding device for holding a sample container while a sample is withdrawn from the sample container.
Another object of the invention is to provide a rack for supporting a plurality of gel tubes for electrophoresis separation, wherein the rack includes a guide for guiding a pipette to a gel tube.
Still another object of the invention is to provide a rack for supporting a plurality of gel tubes, where the rack includes a top and bottom wall defining a chamber, a top wall having a plurality of inlets having a guide surface, and the bottom wall having a plurality of openings with a guide surface aligned with a respective inlet in the top wall for guiding a pipette through said chamber to a respective gel tube.
A further object of the invention is to provide a rack for supporting a plurality of gel tubes for electrophoresis separation including a pair of electric contacts received in a pair of complementary recesses in a gel tank for positioning the rack in a predetermined location in said tank.
Another object of the invention is to provide an electrophoresis separation apparatus having a computer for controlling electric power supply to the gel tanks and for the acquisition of run data for quality control.
The foregoing objects and advantages of the invention are basically attained by providing 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 electrophoretic gel and being supported by the rack. Each of the tubes has a first open end and second open end. The rack includes a chamber for containing a first buffer solution and is in communication with the first end of the tubes. The tank is dimensioned for containing a second buffer solution in contact with the second end of the tubes. An electrical power source is connected to a first electrode in the chamber for contacting the first solution and a second electrode in the tank for contacting the second solution. The apparatus contains
Anderson N. Leigh
Goodman Jack
McGrath Andrew
Large Scale Proteomics Corporation
Roylance Abrams Berdo & Goodman L.L.P.
Starsiak Jr. John S.
Warden Jill
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