Chemistry: electrical and wave energy – Processes and products – Electrostatic field or electrical discharge
Patent
1992-03-27
1994-05-31
Niebling, John F.
Chemistry: electrical and wave energy
Processes and products
Electrostatic field or electrical discharge
C25B 700
Patent
active
053166388
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention concerns treatment of carbohydrates, for analytical and other purposes.
BACKGROUND TO THE INVENTION
International Publication No W088/10422 discloses, inter alia, techniques for analysing carbohydrate structures or distinguishing or separating carbohydrate substances, involving applying carbohydrate substances to an electrophoretic gel and running the gel to cause differential migration of different substances. The carbohydrate substances may be labelled, e.g. with a fluorescent labelling reagent, to impart a charge to the substance, thereby to enable electrophoretic separation, and to enable visualisation of the substances after running of the gel. Visualisation may be effected with the naked eye, but enhanced sensitivity is obtained by viewing with a charge coupled device (CCD).
The present invention concerns a development of such techniques in the form of a further processing step applicable to electrophoretically separated carbohydrate units.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided a method of treating carbohydrate substances, comprising transferring onto a porous membrane by a blotting technique carbohydrate substances labelled with a charged fluorescent labelling reagent which have been run on an electrophoretic gel.
The method enables recovery of charged carbohydrate substances from a gel in a form that is well suited to further treatment or storage, providing a permanent record of electrophoretic separation in the gel. For example, blotted carbohydrate substances can be analysed by probing the blot on the membrane with labelled probes, for instance lectins, sugar specific monoclonal antibodies, viral receptors or other protein probes. Suitable probing techniques are known to those skilled in the art.
The transfer step can be effected using blotting techniques such as are known for blotting proteins from electrophoretic gels, e.g. as discussed in the paper by Jackson and Thompson in Electrophoresis, 1984, 5, 35-42. It is preferred to use an electrotransfer or electroblotting technique, e.g. using semi-dry blot transfer apparatus, which involves contacting the gel with a suitable membrane and applying an electric field to cause resolved materials in the gel to be transferred to the membrane.
A wide range of porous membranes suitable for use in such techniques are commercially available, and are made of materials including activated paper, nitrocellulose, nylon, and polyvinyl difluorone (PVDF), and charged derivatives of such materials. A suitable membrane for use in a particular system can be selected by experiment.
In a further aspect of the invention provides a method of treating carbohydrate substances, comprising applying carbohydrate substances labelled with a charged fluorescent labelling reagent to an electrophoretic gel; running the gel to cause differential migration of different substances; and transferring the substances from the gel onto a porous membrane by a blotting technique.
The gel preferably comprises a relatively dense polyacrylamide gel, having a concentration in the range 15% to 60%, preferably 20% to 40%, although in some cases it may be possible or preferable to use gels of lower concentration.
The gel may be either of uniform concentration or in the form of a gradient gel.
The gel is preferably cross linked, e.g. with N,N' methylenebisacrylamide (bis).
The presently preferred gel comprises a linear polyacrylamide gradient gel having a polyacrylamide concentration (w/v) varying in a continuous gradient from 20% (top) to 40% (bottom). The gel is crosslinked with bis, at a concentration (w/v) varying from 0.53% at the lowest concentration of polyacrylamide to 1.06% at the highest concentration of polyacrylamide. Alternatively, the electrophoretic system described in Neville, Jr., D. M., J. Biol. Chem. 1971, 246, 6328-6334 has been found to give good results.
For good sensitivity the gel is preferably run using a stacking buffer system (also known as moving boundary electrophoresis, m
REFERENCES:
patent: 4589965 (1986-05-01), Kreisher
patent: 5019231 (1991-05-01), Brandley et al.
Turnbull and Gallagher, "Oligosaccharide mapping of heparin sulphate by polyacrylamide-gradient-gel electrophoresis and electrotransfer to nylon membrane," Biochem. J. 251:597-608 (1988).
Lutsik et al., "The use of lectins in the study of human red blood cell membrane glycoproteins," CA 108(11):91259d.
Stocker, et al., "Characterization of Biotin-Labeled Proteoglycans by Electrophoretic Separation on Minigels and Blotting onto Nylon Membranes prior and after Enzymatic Digestion", Analytical Biochemistry 179:245-250 (1989).
Hakim & Lindhardt, "Isolation and recovery of acidic oligosaccharides from polyacrylamide gels by semi-dry electrotransfer", Electrophoresis 11:23-28 (1990).
Peter Jackson, "The use of polyacrylamide-gel electrophoresis for the high-resolution separation of reducing saccharides labelled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid", Biochem. J. 270:705-713 (1990).
Jackson & Thompson, "The immunodetection of brain proteins blotted onto nitrocellulose from fixed and stained two-dimensional polacrylamide gels", Electrophoresis 5:35-42 (1984).
Neville, David M., "Molecular Weight Determination of Protein-dodecyl Sulfate Complexes by Gel Electrophoresis in a Discontinuous Buffer System", Jour. of Biol. Chem. 246:6328-6334 (1971).
Laemmli, U. K., "Clevage of structural Proteins during the Assembly of the Head of Bacteriophage T4", Nature 227:680-685 (1970).
Delacroix-Muirheid Cybille
Niebling John F.
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