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.

1 . An apparatus for conducting two dimensional gel electrophoresis of a protein sample comprising: an isoelectric focusing apparatus, wherein said isoelectric focusing apparatus removably fits into an isolating manifold apparatus designed to accept both said isoelectric focusing apparatus and an apparatus for conducting the second SDS-PAGE separation, and said isolating manifold fits into any standard “mini-gel” electrophoresis apparatus; and an apparatus for conducting the second SDS-PAGE separation step of a two-dimensional gel electrophoresis; said apparatus for conducting the second SDS-PAGE separation also removably fitting into said isolating manifold apparatus. 
     
     
         2 . An apparatus for conducting two dimensional gel electrophoresis of a protein sample comprising:
 a) a first isoelectric focusing apparatus for conducting a two-dimensional gel electrophoresis of a sample further comprising:
 1) a semi-rigid planar sheet having a front and a back surface, a plurality of perforations disposed longitudinally in one direction across said support; and also having a plurality of registration holes disposed longitudinally in the same direction and adjacent to said perforations across said semi-rigid planar sheet; 
 2) a plurality of rectangular holes which defining a casting space for a strip of first dimension electrophoresis gel, and said casting space allowing electrical conductivity for said first dimension electrophoresis gel through said semi-rigid planar sheet to one or more electrolyte reservoirs; said rectangular holes also disposed longitudinally in same direction and adjacent to said perforations and registration holes across said semi-rigid planar sheet; 
 3) at least one surface of said semi-rigid planar sheet treated in such a manner to provide a continuous or discontinuous hydrophilic reactive surface capable of binding an electrophoresis gel polymer by covalently integrating into said gel polymer matrix; 
 4) said perforations and registration holes are sufficient to allow individual electrophoresis gel strips set within said rectangular holes to be separated from the remainder of the semi-rigid planar sheet; 
   B) an isolating manifold apparatus for conducting two-dimensional gel electrophoresis of a protein sample further comprising:
 1) a manifold constructed of elastomeric materials, said manifold having a mounting face and an opposite face and being capable of being fit into a support stand or clamped to a standard gel electrophoresis clamp or glass or plastic plate; 
 2) said manifold provided with a plurality of parallel gel channels, on the opposite face of the manifold, said gel channels having a top and bottom end, and wherein said channels have approximately the same dimension and spacing as the rectangular holes in the semi-rigid planar sheet of  claim 1 , and said gel channels have a depth which defines a space for the first dimension electrophoresis gel strips with a very small laminar void running the entire length of the gel channel when said gel strips are fully hydrated; 
 3) the opposite face of said manifold is further provided with a sufficient number of registration tabs between the gel channels along the perforation line that push through the corresponding registration holes of said semi-rigid support of  claim 1 , such that each gel channel is elastomerically sealed from the others and from outside the manifold, and said opposite face also having a ridge disposed around the perimeter of said manifold, wherein said ridge contacts said semi-rigid support, and holds said semi-rigid support tightly against the manifold face by pressure; 
 4) in the opposite face of said manifold, at the extreme top and bottom of each gel channel, a plurality of ports are also provided which are approximately conical in shape and roughly corresponding in dimensions to a disposable microtiter pipette tip capable of holding volumes around 2 to 200 μL, and said ports are oriented so that when the manifold is mounted on a support stand they are approximately vertical, and said ports having a top and a bottom and the bottom of each port is connected via a collapsible channel to a corresponding gel channel, and said collapsible channel can be either molded into the manifold at the time the manifold is constructed or can be cut through the port after manifold construction, and said collapsible channel is capable of remaining closed and forming a tight fluid seal between the gel channel and the port until a pipette tip or similar means is inserted into the port and allows the collapsible channel to open and creates a fluid path between the port and the gel channel until the tip is removed and said collapsible channel recloses; 
 5) said manifold is capable of being attached to a buffer reservoir means of the type commonly used in gel electrophoresis or mini-gel electrophoresis, wherein the slot of the semi-rigid sheet of  claim 1  can be aligned such that filling of the buffer reservoir means allows electrical conduction of current through each gel strip in said manifold for electrophoresis of samples; 
   C) an apparatus for conducting the second SDS-PAGE separation step of a two-dimensional gel electrophoresis of a sample further comprising:
 1) a molded cassette capable of containing pre-cast gel electrophoresis media, wherein said cassette has a top, two lateral sides and a bottom, and said cassette also has a front plate located on the front of the cassette, and a back plate located on the opposite side of the cassette, both front and back plates having the general dimensions that are known for existing “mini-gel” cassettes; 
 2) said front plate being capable of accepting the mounting of a first dimension gel strip within a horizontally oriented port or opening which is molded into the surface of said front plate, and wherein said horizontally oriented port or opening within the front plate is capable of being temporarily sealed flush with the Inside face of the front plate; 
 3) said cassette having sealing spacers provided at the lateral sides and bottom which forms an interior laminar space within the cassette between the front and back plates capable of containing gel electrophoresis media in either a polymerized or an unpolymerized state within said space, and wherein said cassette is capable of maintaining this interior laminar space indefinitely; 
 4) said front and back plate further comprised of glass or molded rigid plastic or elastomeric materials compatible with aqueous solutions and said back plate also being capable of having the gel facing surface of said back plate modified so that polymerized gel electrophoresis media will chemically bond with the gel facing surface of said back plate during or after polymerization; and 
 5) the horizontally oriented port or opening within the front plate further comprises a tight fitting elastomeric seal forming a water-tight barrier in said horizontally oriented port or opening, wherein said seal is comprised of molded rigid plastic or elastomeric materials compatible with aqueous solutions, and said seal provides the attachment of a first dimension gel strip with its backing into a tight-fitting groove on the gel face of the front plate, and said seal has a tab allowing for the removal of the seal after the first dimension gel strip is in contact with the second dimension gel media exposed through said horizontal opening when said seal is in place. 
   
     
     
         3 . A method for conducting two-dimensional gel electrophoresis on a sample using the apparatus of  claim 1  comprising the steps of:
 a) partially dehydrated pre-cast polyacrylamide pH gradient gel strips bound to a perforated semi-rigid support or backing are pressed or mounted into molded channels on one side of said elastomeric manifold so that each strip is sealed with a laminar space above said gel, each laminar space is accessible for filling through closable ports on top and bottom on the side of the elastomeric manifold opposite from the strips; 
 b) the elastomeric manifold is mounted at an angle on a support stand wherein samples containing proteins to be focused are loaded through the bottom ports through opening the closable channels and filling the laminar space above each strip, and the top ports are forced open so that the laminar space can be vented; 
 c) samples loaded are incubated for sufficient time so as to re-hydrate into the isoelectric focusing gel media; 
 d) the manifold with up to eight attached sample isoelectric focusing strips is mounted adjacent to the buffer core and sealed, a buffer dam or a second manifold is installed on the opposite side of said buffer core and said manifold or manifolds with said buffer core is dropped into the lower buffer chamber; 
 e) electrolytic buffer solutions are added to the reservoirs to a level above connecting slots punched through the perforated semi-rigid backing at the top and bottom of each gel strip allowing an electrical path to the isoelectric focusing gel from the reservoirs; 
 f) the apparatus is attached to a constant voltage power supply and run for a sufficient period of time to migrate the samples into the gel media and then the voltage is increased and continued until the samples are sufficiently focused within the gel media; 
 g) the voltage is removed and said manifold is remounted on said support stand, a re-equilibration solution is the added to the top ports of the laminar spaces of each first dimension isoelectric focusing strip and allowed to fill said spaces by gravity means, during addition of solution, the lower ports of the laminar spaces are kept open to provide venting; 
 h) upon completion of filling of said laminar spaces and waiting a sufficient time to denature the samples in the gel media, said strips are removed from the manifold and separated from each other by applying opposing force upon said perforations in between each strip; 
 i) a separated gel strip is then pressed into an elastomeric seal means with the gel side on the opposite side from the seal, and the strip and sealing means are pressed into a slot in the lateral side of the second dimension gel cassette so that the gel strip surface is in contact with SDS-PAGE gel media of the second dimension gel cassette in the slot; 
 j) one or more second dimension gel cassettes are then placed against a buffer core of the mini-gel electrophoresis apparatus and kept in place by pressure through the elastomeric seal around the bottom and both sides of the buffer core, and the buffer core and lower buffer chamber are filled with electrolyte solution, and the system is energized to apply either constant current or constant voltage; 
 k) after a period of time sufficient to migrate the protein samples into the second dimension gel, the system is de-energized and the first dimension strip and elastomeric seal are removed from the second dimension gel cassette, the system is energized for sufficient time to separate the sample proteins on the second dimension gel; and 
 l) upon completion, the system is de-energized and the second dimension gel cassette is then removed from the apparatus and separated from the buffer core where it is further processed.