Chemistry: electrical and wave energy – Processes and products – Electrostatic field or electrical discharge
Patent
1987-09-14
1989-10-17
Niebling, John F.
Chemistry: electrical and wave energy
Processes and products
Electrostatic field or electrical discharge
B01K 500
Patent
active
048744913
DESCRIPTION:
BRIEF SUMMARY
The present invention is concerned with the field of electrophoresis and relates more particularly to a method of supplying buffer solutions to electrophoretic separation procedures by means of using buffer-containing gels as buffer reservoirs, thus eliminating the need for special buffer vessels in equipments employed for electrophoresis.
Electrophoretic separation is often carried out in a stabilizing matrix such as e.g. an agarose or polyacrylamide gel. The matrix is usually placed on a support in the form of for instance a disc of plastics, whereupon two opposite sides of the matrix are connected to one electrode buffer each. In case the matrix is enclosed within a cassette of for instance glass or plastics the contact is nevertheless made feasible due to the cassette being open at two opposite sides. The electrodes may be inserted in vessels containing the electrode buffers, and the connection to the matrix is established by means of some type of liquid bridge, for example a strip of paper or some other material capable of becoming soaked with buffer.
When electrophoretic separation is carried out in systems of this type a sample volume is deposited on the gel matrix, and then an electric field is applied between the electrodes whereby charged components of the sample are caused to migrate in the gel matrix. Experimental conditions may vary to a great extent--e.g. as regards the choice of gel matrix, pore size to thus permit separation also with respect to molecule size or particle size, or as regards regulation of pH and correspondingly regulation of the charges on the sample components. A feature common to all of these techniques is that the migration of the components in the gel matrix proceeds in an electrolytic medium.
With respect to the anodic buffers and cathodic buffers employed, an important requirement is that they must have a high buffering capacity; that is, they must not undergo any substantial change during the electrophoretic procedure, in spite of the reactions occurring at each of the electrodes. The most significant factors determining buffering capacity are the types of buffer components chosen, their concentrations, and the volume of the buffer solution.
As mentioned above electrophoresis is conventionally carried out with an equipment comprising special buffer tanks each of which is filled with the desired volume of its respective buffer. However, in the cases of some applications it is difficult to employ solutions of high concentration, because of liquid flow arising due to capillary forces at for instance the edges of the gel matrix. By means of maintaining a low electrode buffer concentration it is possible to avoid such faultiness or downright short-circuiting in the electric field of the separation gel as might otherwise be caused by the said type of liquid flow. A low electrode buffer concentration may be compensated for by increasing the buffer volume, although this of course necessitates incorporation of sufficiently large buffer vessels in the electrophoretic equipment. The potential drop outside the separation matrix could however in such cases be considerable. Especially in systems working with comparatively small gel matrices, for example in more or less automated systems, handling of the buffer solutions involves practical problems; it is desirable, therefore, that one should be able to reduce handling of liquid volumes to the greatest possible extent.
U.S. Pat. No. 3,715,295 has identified this problem and suggests mixing the buffer with a substance by means of which the buffer is obtained in a "semisolid" form. According to a preferred embodiment the buffer is incorporated in silica particles, whereupon a semisolid substance is added. Irrespective of the type of "semisolid" form chosen, a suitable volume thereof is transferred to a well at each end of the supporting baseplate of the electrophoretic equipment; thereafter the separation gel is positioned on the plate so as to establish direct liquid contact between the separation gel and the buffer. It should be not
REFERENCES:
patent: 3062731 (1962-11-01), Durrum
patent: 3715295 (1973-02-01), Tocci
patent: 4006069 (1977-02-01), Hiratsuka
Andrews, Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications, 2nd ed., p. 148 (1986).
Niebling John F.
Pharmacia AB
Philpitt Fred
Rodriguez Isabelle
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