Chemistry: electrical and wave energy – Processes and products – Electrostatic field or electrical discharge
Patent
1987-09-29
1989-07-04
Niebling, John F.
Chemistry: electrical and wave energy
Processes and products
Electrostatic field or electrical discharge
2041828, 2041829, 204299R, C25D 112
Patent
active
048447820
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to an improved method in the after-treatment of electrophoretic gels containing separated components, in that the gel is caused to rotate in a selected fixation, staining or destaining solution with continuous temperature measurement during at least a part of the total processing time calculated from given processing data.
Electrophoretic separation in a supportive phase in the form of a gel medium such as agarose or polyacrylamide, is usually performed by applying a sample to a very small area of the gel which contains an electrolytic liquid medium. Whereupon an electric field is applied over the gel. Then during a given period of time different components or different groups of components will migrate along distances of different lengths, depending on the experimental conditions such as the choice of buffer medium, pH and concomitantly of the charges on the sample components, or the choice of pore size in the gel matrix. When the electric field is removed and the separation is thus discontinued, the components of the sample will be found at different places along the migration path, often in the form of more of less distinct bands. Such a concentration of material to narrow bands is of paramount importance for subsequent analysis of the separation result; and special techniques have been developed for obtaining particularly sharp bands, for example isoelectric focusing.
Since the concentration of material in the bands is a high one as compared to their surroundings, diffusion from the bands into the surrounding gel regions will start as soon as the electric field is removed. Therefore, the first step performed after the separation will often be a treatment of the gel with a solution containing substances that will fix the bands to thereby prevent diffusion.
After the fixation step the position of the sample components have to be located on the gel for qualitative and/or quantitative analysis. In cases where the sample has originally contained e.g. fluorescent or radioactively labeled components detection can be carried out directly with the aid of a suitable detector; in most cases, however, the bands are subjected to staining for being rendered visible. This involves contacting the gel with a solution which contains dyes which will bind to the sample components, but not to the gel matrix. In this step it is an important requirement that staining be uniform, as otherwise a quantitative evaluation would be hampered by much uncertainty; consequently the supply of dye to the gel surface must be uniform, with ample time being allowed for the dye to bind to the components involved. The time actually required will depend on gel thickness, stirring rate, concentration of staining solution, and the temperature prevailing. According to the present state of the art staining is usually carried out by immersing the gel in a staining bath, and the bath is given a shake now and then. In order to ensure complete staining it is often preferred to leave the gel in the bath for a relatively long time, e.g. overnight.
During the staining step the dye will thus diffuse into the whole gel matrix, and for this reason it is necessary to wash off non-bound dye in a special so-called destaining bath. When the gel has been treated in this manner the bands will show up, and their positions, areas and colour intensities can be determined for a final evaluation.
Thus after the electrophoretic separation the gel is to be subjected to a plurality of treating steps: (1) fixation, (2) staining, and (3) washing for removal of non-bound dye, before evaluation is initiated. This sequence is customarily carried out by means of transferring the gel from container to container, the whole procedure taking usually at least six hours, the most common practice being actually to extend e.g. the staining step over one night. The containers may be provided with stirring means by which the solution is circulated in the container; but more often the container is simply subjected to shaking now and again, as mentioned abov
REFERENCES:
patent: 4222843 (1986-09-01), Suzuki
patent: 4254084 (1981-03-01), Blum
patent: 4612106 (1986-09-01), Kromer
Analytical Biochemistry, vol. 112, No. 2, 1981, pp. 232-235, Terranova, A. C., "A Rotating Temperature-Controlled Water Bath for Isozyme Development in Polyacrylamide Slab Gels".
Electrophoresis, vol. 6, No. 8, 1985, pp. 408-09, Birgit C. et al., "Protein Staining of 6mm Diameter Sodium Dodecyl Sulfate-Polyacrylamide Gels Within One and a Half Hours".
Hagerlid Peter K.
Hitchman Michael
Jacobson Gunilla M. C.
Wheeler Richard
Niebling John F.
Pharmacia AB
Philpitt Fred
Rodriguez Isabelle
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