Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Patent
1995-07-18
1997-01-14
Valentine, Donald R.
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
204462, 204464, G01N 2726
Patent
active
055935613
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a multiple electrophoresis method and apparatus for migration and transfer of macromolecules, such as proteins or nucleic acids.
It is now well known to separate samples of macromolecules by electrophoresis, causing them to migrate through a plate of gel under the effect of an electric field. The samples of macromolecules travel through the gel over distances that are a function of their molecular masses. Under the effect of a second electric field, perpendicular to the first and to the plates of gel, it is possible to transfer the macromolecules onto microporous membranes associated with the plates of gel.
That technique, which was initially lengthy and difficult to implement, has been improved and simplified little by little so that it is now possible to cause macromolecules to migrate and separate and then to transfer them, all within the same vessel and using the same network of electrodes, without any manipulation of the plates of gel or the membranes between migration and transfer of the macromolecules.
In particular, international application WO 90/02601 in the name of the Applicant describes a multiple electrophoresis vessel in which it is possible to place a relatively large number of plates of gel associated with membranes, in order to perform separation and transfer of macromolecules in a manner that is automatic or quasi-automatic. In addition, French patent application 91 08578 in the name of the Applicant describes cassettes for making and manipulating plates of gel associated with membranes, and suitable for use in the electrophoresis vessel described in the above-mentioned international application.
Although those known means have made it possible to achieve results that are quite remarkable, it remains relatively difficult to make up the units each comprising a plate of gel plus a membrane, and special care is required to ensure that the plates of gel are uniform and do not contain bubbles of air, and to ensure that the membranes associated with said plates of gel are thoroughly plane and adhere over their entire working area to the plates of gel. Also, because the gel ages quickly and very poorly, particularly when it is agarose, it is impossible to prepare large quantities of plates of gel with membranes a long time in advance and to store them for subsequent use.
A particular object of the present invention is to provide a solution to the problems that is simple, effective, and cheap.
The invention provides a multiple electrophoresis method and apparatus for separation of macromolecules in gel and transfer thereof onto membranes, but without using prefabricated gel-membrane units.
The invention thus provides a multiple electrophoresis method for controlled migration of macromolecules and transfer thereof onto membranes in a vessel containing a plurality of parallel elongate electrodes and having means for applying electrical potentials to the electrodes for establishing between them successively a first electric field for macromolecule migration, and then a second electric field perpendicular to the first for transferring the macromolecules onto the membranes, the method being characterized in that it consists in placing in the vessel membranes which are parallel to one another and to the electrodes and which extend between the electrodes, and in filling the vessel with an electrophoresis gel in the liquid phase, the filling being performed before or after the membranes have been placed in the vessel, then in solidifying the gel, in subsequently placing samples of macromolecules in wells formed in the gel along one of the edges of the membranes, in applying electrical potentials to the electrodes initially to cause the macromolecules to migrate through the gel, and then to transfer them onto the membranes, then in liquefying, dissolving, or decomposing the gel, and in removing the membranes from the vessel.
The invention thus makes it possible to avoid prefabricating plates of gel before performing electrophoresis, thus achieving a considerable saving in time, m
REFERENCES:
patent: 3888758 (1975-06-01), Saeed
patent: 4994166 (1991-02-01), Fernwood et al.
Cognard Dominique
Hache Jean
Bertin & Cie
Noguerola Alexander
Valentine Donald R.
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