Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Patent
1995-10-10
1997-07-22
Tung, T.
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
204519, 204450, 204456, 204600, 204606, 204607, 204457, 204608, G01N 2726
Patent
active
056500556
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates Co a method of, and apparatus for, preparative electrophoresis and, in particular, to such methods and apparatus useful in large scale recovery of macromolecules such as proteins.
DESCRIPTION OF PRIOR ART
Electrophoretic separation of macromolecule such as proteins, peptides, nucleic acids and Other compounds is typically carried out using a medium such as a chemical gel or a membrane. The process involves bringing a first buffer containing a mixture of macromolecule species into contact with the electrophoretic medium whilst an electrophoretic potential is applied along or across the medium so that at least some of the macromolecules are induced to migrate into the medium. The medium typically has a cut-off pore size which is Selected so as to allow the entry of molecules having less than a given size. Further, manipulation of the pH of the buffers used in the separation process can contribute to the selection of the molecule species to be separated. The separated molecules are normally caused to emerge from the medium into a second buffer for collection.
A known problem with such electrophoretic separation method is that molecules larger than the given size will foul the surface of the electrophoretic medium.
This problem has been overcome by a technique of intermittently reversing the polarity of the electrophoretic potential so as to draw the fouling molecules away from the surface of the medium whilst ensuring that there is Still a Positive movement of the molecules to be separated towards the second buffer. To achieve this, the polarity reversal is typically conducted in short bursts only and the total time of polarity reversal is necessarily a fraction of the time of applying the "forward" polarity of the electrophoretic potential (see Australian Patent specification 601040).
Typically, the reversal of the polarity is carried out repeatedly at a rate such that the membrane is constantly being cleared of any surface fouling material. As such, it will be appreciated that the polarity reversal process is analogous to vibrating a sieve. In this model all undersize particles Pass through the sieve but oversize particles clog it. Vibrating the sieve clears the oversize particles away from The surface of the sieve and allows more undersize particles to pass through it.
The known electrophoretic separation methods are generally effective for separating macromolecules having different transport Characteristics (i.e. size, charge, shape, isoelectric point) which have actually entered the electrophoretic medium. Where, however, the transport characteristics of two or more of the macromolecules in the medium are similar, it can be difficult to achieve a complete separation of one of the macromolecular species in the mixture from the others using such conventional methods.
The extent to which two molecule species can be physically separated in an electrophoretic medium will depend not only on their charge characteristics but also on the mean length of paths that the molecules may follow through the electrophoretic medium. Even for a very long mean path length there may not be complete separation of the molecule species due to molecules of The faster moving species being trapped in the medium and contaminating the slower moving molecular species. Even if merely lengthening the medium would achieve complete separation of two species that have similar transport characteristics, the use of such an elongate electrophoretic medium would be inconvenient and there may be practical problems to overcome, such as heat dissipation.
In contrast, the present method and apparatus utilizes the observed differential in the rate of progress of components using a selected electrophoretic medium and/or pH of the buffer so that different species of macromolecules can be conveniently and generally completely separated from one another despite similarities in their transport characteristics.
SUMMARY OF THE INVENTION
The present invention provides a method for the electrophore
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pp. 116 of Webster's II New Riverside University Dictionary, Riverside Publishing Company 1994.
Noguerola Alex
Striker Michael J.
Tung T.
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