Stock material or miscellaneous articles – Structurally defined web or sheet – Discontinuous or differential coating – impregnation or bond
Patent
1989-08-21
1997-09-30
Lee, Helen
Stock material or miscellaneous articles
Structurally defined web or sheet
Discontinuous or differential coating, impregnation or bond
428252, 428253, 428286, B32B 0700
Patent
active
056724160
DESCRIPTION:
BRIEF SUMMARY
The invention relates to new fabrics with improved properties for the preparation of electrophoresis gels and processes for producing them.
Polyacrylamide gels and agarose are used in numerous electrophoresis techniques as anticonvective and screening matrices. The gels are used in two forms: particularly preferred for first-dimension separation in two-dimensional electrophoresis; and nowadays, namely sodium dodecylsulphate (SDS) electrophoresis and isoelectric focussing.
A fact common to both configurations is that during the separation the gels are supported from the outside, e.g. by the walls of a glass test tube or a sheet of glass. After separation the gels are evaluated, e.g. by protein staining, enzyme visualisation or blotting. The unsupported gels are subjected to considerable mechanical stress, which means that gels with a layer thickness of less than 0.7 mm cannot be used.
A further considerable disadvantage is that conventional gels are not dimensionally stable and will readily change their dimensions by swelling or shrinkage in solvents of different composition, making it difficult or impossible to evaluate the components visualised.
By polymerising the gels onto suitably pretreated polyester films it is possible to overcome to some extent the disadvantages described above. Using suitably pretreated polyester films it is possible to prepare ultrathin gels, i.e. those with a thickness of about 50-350 microns, which have crucial practical advantages over gels of conventional thickness (e.g. 1 to 3 mm) in isoelectric focussing and SDS electrophoresis. However, film-supported gels of this kind have the disadvantage that they are not optimal for capillary blotting and are completely unsuitable for electroblotting.
An alternative to the externally stabilised gels are fabric-supported gels with internal stabilisation. Such gels are open on both sides and are therefore particularly suitable for blotting. However, they are also of interest for other applications, e.g. preparative isoelectric focussing. Contrary to expectations, however, it has been found that such electrophoresis gels prepared on fabrics do not meet the practical requirements.
Hitherto, it has been assumed that because of the greater contact surface between the fabric and the gel, as compared with gels on support films, the fabric-supported gels will adhere adequately even to non-pretreated fabrics. This may be true for those applications in which the fabric-supported gels are merely stained after electrophoretic separation, since no very great demands are made of the gel adhesion in such cases. However, the situation is different in the application of fabric-supported gels which is currently of most importance, namely blotting. In this technique, after separation from the gel using suitable membranes, e.g. nitro-cellulose membranes, a blot is produced in which the separated components are immobilised so that they are then available for further reactions, e.g. immunological detection methods. The transfer of the separated components into the membrane requires good contact between the surface of the gel and the nitrocellulose membrane since otherwise some of the resolution achieved in the gel will be lost.
The transfer may be carried out either by capillary blotting or by electroblotting in an electrical field. In both transfer techniques, pressure is applied to the gel and the membrane in order to obtain as accurate a copy as possible in the blot.
For further evaluation of the blot the membrane must be lifted away from the surface of the gel. It has been found, unexpectedly, that the adhesion of the gels to the supporting fabric is inadequate. Parts of the gel or, in some cases, the entire surface of the gel is or are pulled away from the membrane as well, so that subsequent evaluation of the blot is impaired or even rendered completely impossible.
A further disadvantage of the fabric-supported gels used hitherto is that these gels frequently cannot be totally destained and consequently they have an uneven background. This may have a d
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Fabric Reinforced Polyacrylamide Gels For Electroblotting --H. Nishizawa et al., Electrophoresis vol. 6, pp. 349-350 (1985).
Demharter Manfred
Radola Bertold
Schwall Horst
Devlin M-E. M.
Lee Helen
Raymond R. P.
Serva Feinbiochemica GmbH & Co.
Stempel A. R.
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