Claims
- 1. A method of separating and analyzing saccharides within a mixture of saccharides, comprising the steps of:
- reacting the saccharides in the mixture with charge-generating moieties and moieties capable of fluorescing in order to form saccharide conjugates;
- subjecting the saccharide conjugates to electrophoretic separation in a gel;
- electro-blotting the separated saccharides in the gel to the surface of a support membrane; and
- contacting labeled probes with the membrane and determining binding affinity of the probes to saccharides on the membrane.
- 2. The method as claimed in claim 1, wherein the charge-generating moieties and moieties capable of fluorescing are the same moieties which are charge-generating moieties capable of fluorescing.
- 3. The method as claimed in claim 2, wherein the charge-generating moieties capable of fluorescing are selected from the group consisting of:
- 1-amino-4-(2-hydroxyethyl)piperazine;
- 2-amine-9-hydroxyfluorene;
- 2-amino-6-hydroxy-8-mercaptopurine;
- 4-amino-6-hydroxy-2-mercaptopyrimidine monohydrate;
- 2-amino-4-hydroxy-6-methylpyrimidine;
- 4-amino-3-hydroxy-1-naphthalenesulfonic acid;
- 4-amino-6-hydroxy-1-naphthalenesulfonic acid;
- 6-amino-4-hydroxy-2-naphthalenesulfonic acid monohydrate;
- 7-amino-4-hydroxy-2-naphthalenesulfonic acid monohydrate;
- 3-amino-5-hydroxypyrazole;
- 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine;
- 4-amino-1-naphthalenecarbonitrila;
- 3-amino-2,7-naphthalenedisulfonic acid, monosodium salt;
- 7-amino-1,3-naphthalenedisulfonic acid, monopotassium salt;
- 2-amino-1-naphthalenesulfonic acid;
- 4-amino-1-naphthalenesulfonic acid;
- 5-amino-2-naphthalenesulfonic acid;
- 8-amino-2-naphthalenesulfonic acid;
- 4-amino-1,8-naphthalic anhydride;
- 4-amino-1,8-naphthalimide;
- 3-amino-2-naphthoic acid;
- 4-amino-1,2-naphthoquinone hemihydrate;
- 6-aminonicotinamide;
- 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid, sodium salt; and
- N-(2-aminoethyl)-4-amino-3,6-disulfo-1,8-naphthalene, dipotassium salt
- and any salts of the acids and acids of the salts.
- 4. The method as claimed in claim 2, wherein the charge-generating moieties capable of fluorescing have a molecular weight of less than 500 and include an amino group.
- 5. The method as claimed in claim 1, wherein the charge-generating moieties are selected from the group consisting of sulfonates and carbonates.
- 6. The method as claimed in claim 1, wherein the moieties capable of fluorescing are selected from the group consisting of: naphthalene, fluorescein, aminonaphthalene and amino fluorescein.
- 7. The method as claimed in claim 1, wherein the mixture of saccharides is a mixture of heparin fragments obtained from partial nitrous acid digests.
- 8. The method as claimed in claim 1, wherein the saccharide mixture includes tetrose derived from asialo-GM1.
- 9. The method as claimed in claim 1, wherein the mixture of saccharides includes neutral oligosaccharides.
- 10. The method as claimed in claim 9, further comprising:
- coating the surface of the membrane having the saccharides electro-blotted thereon with polyisobutylene methylmethacrylate.
- 11. The method as claimed in claim 1, wherein the mixture of saccharides includes saccharides selected from the group consisting of mono-, di- and trisaccharides.
- 12. The method as claimed in claim 1, wherein the labeled probe is a labeled protein probe.
- 13. A method of resolving a mixture of saccharides into distinct bands of saccharides which are closely related or identical, comprising the steps of:
- reacting the mixture of saccharides with charge-generating moieties and moieties capable of fluorescing to form saccharide conjugates;
- subjecting the conjugates to gel electrophoresis separation by the application of an electrical field for a sufficient period of time to form the separate bands in the electrophoresis gel;
- transferring the separate bands of saccharides in the electrophoresis gel onto a membrane surface; and
- exposing the membrane surface to ultraviolet light to determine the bands of saccharides.
- 14. A method of separating a mixture of carbohydrate, comprising the steps of:
- reacting the carbohydrate mixture with charge-generating moieties capable of fluorescing to form conjugates;
- subjecting the conjugates to electrophoretic separation in a gel to provide separate bands of conjugates, wherein the charge-generating moieties capable of fluorescing are selected from the group consisting of monosulfonic and disulfonic acid derivatives of amino naphthalene.
- 15. The method as claimed in claim 14, wherein the charge-generating moieties capable of fluorescing are alpha-amino naphthalenes.
- 16. The method as claimed in claim 14, further comprising:
- transferring the bands out by electro-blotting the bands in the gel onto the surface of a membrane.
- 17. The method as claimed in claim 16, wherein the carbohydrate mixture comprises saccharides selected from the group consisting of monosaccharides, disaccharides and trisaccharides.
- 18. The method as claimed in claim 16, further comprising:
- contacting the membrane surface with labeled probes to determine the affinity of the probes to bind to saccharides on the membrane surface; and
- washing away any labeled probes not bound to saccharides on the surface and detecting bound probes by their label.
- 19. The method as claimed in claim 16, wherein the mixture of carbohydrates includes specific saccharides in an amount in the range of 1 to 10 picomoles and wherein such saccharides present in an amount of 1 to 10 picomoles are observable in distinct bands on the membrane surface when viewed under ultraviolet light.
- 20. The method as claimed in claim 19, wherein distinct bands of saccharides on the membrane surface includes saccharides in an amount of from 1 to 5 picomoles.
- 21. A membrane, the surface of which has electro-blotted thereon a plurality of distinct bands of saccharides wherein the saccharides are bound to a charge-generating moiety and a moiety capable of fluorescing.
- 22. The membrane as claimed in claim 21, wherein the charge-generating moiety and moiety capable of fluorescing is the same moiety which is a charge-generating moiety capable of fluorescing.
- 23. The membrane as claimed in claim 22, wherein the charge-generating moiety capable of fluorescing is selected from the group consisting of 4-amino-1-naphthalene sulfonic acid; and 4-amino-1-naphthalene sulfonic acid having one or two additional sulfonic acid groups attached to the naphthalene ring at the 5, 6, 7 or 8 positions of the rings.
- 24. The membrane as claimed in claim 23, wherein the fluorescent label is 4-amino-1-naphthalene sulfonic acid.
- 25. A method of separating and analyzing saccharides within a mixture of saccharides, comprising the steps of:
- reacting the saccharides in the mixture with naphthalene sulfonic acid or a derivative thereof, the reacting being carried out in order to form conjugates;
- subjecting the conjugates to electrophoretic separation in a gel;
- electro-blotting the separated saccharides in the gel onto the surface of a support membrane; and
- contacting labeled probes with the membrane and determining the binding affinity of the probes to saccharides on the membrane.
- 26. A bi-functional conjugate in the form of a carbohydrate having bound thereto a monosulfonic or disulfonic acid derivative of amino naphthalene or a salt thereof.
CROSS-REFERENCE
This application is a continuation-in-part of our pending U.S. application Ser. No. 481,367, filed Feb. 16, 1990, now U.S. Pat. No. 5,019,231, which application is incorporated herein by reference and to which application we claim priority under 35 USC .sctn.120. Further, this application is related in part to two other co-pending U.S. applications filed concurrently with the present application on Sept. 27, 1990. One related application is entitled "Two-Dimensional Electrophoretic Separation of Carbohydrates," invented by Brian K. Brandley and Robert J. Stack; the other application is entitled "Fluorescent Tag for Sugar Electrophoresis," invented by Brian K. Brandley, Michael Tiemeyer and Robert J. Stack, all of whom are co-inventors working in the same research organization as the present inventors with an obligation to assign the invention to the same entity. The above-referenced applications are each incorporated herein by reference in their entireties.
US Referenced Citations (3)
Number |
Name |
Date |
Kind |
4109075 |
Deaton |
Aug 1978 |
|
4305799 |
Scharz et al. |
Dec 1981 |
|
4666581 |
Itoh et al. |
May 1987 |
|
Foreign Referenced Citations (3)
Number |
Date |
Country |
WO8810422 |
Dec 1988 |
WOX |
2175690 |
Dec 1986 |
GBX |
2196734 |
May 1988 |
GBX |
Continuation in Parts (1)
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Number |
Date |
Country |
Parent |
481367 |
Feb 1990 |
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