Facile method and apparatus for the analysis of biological macromolecules in two dimensions using common and familiar electrophoresis formats

Abstract

An ensemble of components and methods are disclosed for utilizing two-dimensional electrophoresis in polyacrylamide or related polymer gels using common, existing and familiar electrophoresis formats and equipment. The disclosed invention makes two-dimensional electrophoresis convenient and easy to use for individuals already using vertical “mini-gel” type systems. The invention discloses the combination of pre-cast disposable gels for both first and second separation dimensions using novel support devices and cassettes that simplify the difficult multiple sample handling and processing steps inherent in ordinary two-dimensional electrophoresis methods. Devices and methods are disclosed in said invention that provide exceptional gel to gel reproducibility.

Description

[0001]This application claims priority from U.S. Provisional Application No. 60 / 147,490 filed on Aug. 9, 1999.TECHNICAL FIELD AND INDUSTRIAL APPLICABILITY OF INVENTION[0002]The invention relates to the production of acrylamide or other gels for use in separations of proteins, nucleic acids or other biological materials. The invention further relates to making two-dimensional electrophoresis convenient and easy to use for individuals already using vertical “mini-gel” type systems. The invention discloses the combination of pre-cast disposable gels for both first and second separation dimensions using novel support devices and cassettes that simplify the difficult multiple sample handling and processing steps inherent in ordinary two-dimensional electrophoresis methods.BACKGROUND OF THE INVENTION[0003]Polyacrylamide gel electrophoresis has become one of the most frequently used techniques for the separation of biological macromolecules such as proteins, nucleic acids and polysaccharid...

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Benefits of technology

[0008]The manifold also provides for a sealed laminar void space running the length of each gel strip adjacent to the face of each partially dehydrated gel, which can be filled with a fluid medium. The fluid medium is adsorbed into the partially dehydrated gel so as to hydrate it. In a preferred embodiment, the manifold is molded from elastomeric or semi-elastomeric polymers including but not limited to polysiloxanes, polyisoprenes, polyisobutylenes and polysulfides. Other elastomeric polymers which have physical properties that-provide for the easy-and tight seal of the semi-rigid support onto the manifold surface would suffice.

[0011]A diagonal support stand is provided in the present invention to hold the gel strip-manifold assembly for the introduction and incubation of said fluid media through the bottom port of each laminar space and to allow for the venting of entrained air through the top port. The 9-mm spacing provides for convenient simultaneous sample loading with a commonly used 8 or 12 place pipettor (Gilson). A sample can then be introduced into the isoelectric focusing gel by adsorption into and re-hydration of the gel while being incubated in the said gel strip-manifold assembly.

[0016]It is often desirable to remove first dimension gel strips or tubes from the second dimension gel after the sample has completely migrated out of the first dimension gel and into the second dimension gel. It is also desirable for the path of the electrical conductivity to bypass the point of contact between the first and second dimension gel. This bypass minimizes streaking from poorly solubilized residual components on the surface of the second dimension gel. Provision is provided for this in the way of a tab or handle means on the horizontal port or seal such that the seal and first dimensional strip can be removed following de-energizing the apparatus. After de-energizing, the removal of the first dimension gel is accomplished without disassembly or emptying of the electrolyte solutions in the reservoirs. Following removal of the seal and first dimension gel, the apparatus is re-energized and the second dimension gel is run to completion with the electrical path running through the now open horizontal port, thus limiting the time without electric power to a minimum.

[0017]Therefore, it is an object of the present invention to provide an apparatus which facilitates two-dimensional gel electrophoresis.

[0018]Another object of the invention is to reduce handling and movement of the gel, reduce operator exposure to buffers and samples and improve test result reliability.

Problems solved by technology

This two-dimensional separation of complex mixtures using polyacrylamide electrophoresis is a powerful but difficult technique.

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