US5035786A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Fluorescent tag for sugar electrophoresis
Assignee: Glycomed Inc
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Filing: 1990-02-16
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Granted: 1991-07-30
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Expires: 2010-02-16
Abstract
Abstract A method of separating mixtures of saccharides into distinct detectable groups is disclosed. The method comprises modifying 1-amino-4-naphthalene sulfonic acid (ANSA) with a light-sensitive azido-group and binding the modified ANSA to saccharides to form ANSA/saccharide conjugates. The conjugates are subjected to electrophorectic separation to obtain separate groups of conjugates in the gel. The groups of conjugates are transferred from the gel to the surface of a membrane which is exposed to light for a sufficient time and light frequency to activate the azido-group. The light-activated azido-groups attached to the surface of the membrane. The ANSA/saccharides conjugates can then be contacted with labeled probes such as radiolabeled proteins to determine the affinity of the probes to particular saccharides.
Inventors
- 👤 Brian K. Brandley
- 👤 Michael Tiemeyer
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 20:44
CPC Classifications
G; G01; G01N; G01N27/00; G01N27/26; G01N27/416; G01N27/447; G01N27/44704; G01N27/44717; G01N27/44721; G01N27/44726
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
110 words
A method of separating a mixture of saccharides into distinct detectable groups, comprising the steps of: reacting the mixture of saccharides with a modified form of 1-amino-4-naphthalene sulfonic acid to form conjugates, the 1-amino-4-naphthalene sulfonic acid being modified by the addition of a light-sensitive azido-group; subjecting the conjugates to gel electrophoresis in an electrophoresis gel for a sufficient period of time to form separate groups of conjugates in the electrophoresis gel; electro-blotting the separate groups of conjugates from the electrophoresis gel onto the surface of a membrane; and subjecting the conjugates on the membrane to light for a sufficient time and of a light frequency which activates the light-sensitive azido-group.
Claim 2
Independent Claim
110 words
A method of separating a mixture of saccharides into distinct detectable groups, comprising the steps of: reacting the mixture of saccharides with a modified form of 1-amino-4-naphthalene sulfonic acid to form conjugates, the 1-amino-4-naphthalene sulfonic acid being modified by the addition of a light-sensitive azido-group; subjecting the conjugates to gel electrophoresis in an electrophoresis gel for a sufficient period of time to form separate groups of conjugates in the electrophoresis gel; electro-blotting the separate groups of conjugates from the electrophoresis gel onto the surface of a membrane; and subjecting the conjugates on the membrane to light for a sufficient time and of a light frequency which activates the light-sensitive azido-group.
Claim 3
Dependent Claim
35 words
The method as claimed in claim 1, further comprising: contacting the separate groups of conjugates on the membrane with labeled probes to determine the affinity of the probes to bind to a group of conjugates.
Claim 4
Dependent Claim
28 words
The method as claimed in claim 2, further comprising: washing away any labeled probes not bound to a group of conjugates and detecting bound probes by their label.
Claim 5
Dependent Claim
16 words
The method as claimed in claim 2, wherein the labeled probe is a labeled protein probe.
Claim 6
Dependent Claim
13 words
The method as claimed in claim 4, wherein the label is a radiolabel.
Claim 7
Dependent Claim
20 words
The method as claimed in claim 4, wherein the labeled protein probe is a labeled antireceptor protein of a virus.
Claim 8
Dependent Claim
26 words
The method as claimed in claim 4, wherein the electrophoresis gel is comprised of about 40% acrylamide, about 5% bis gels, in a Tris-glycine buffer system.
Claim 9
Dependent Claim
28 words
The method as claimed in claim 7, wherein the Tris-glycine buffer system includes approximately 25 mM Tris, approximately 195 mM glycine and has a pH of about 8.3.
Claim 10
Dependent Claim
28 words
The method as claimed in claim 1, wherein the azido-group is bound to the naphthalene ring system of the ANSA at the 5, 6, 7 or 8 position.
Claim 11
Dependent Claim
18 words
A method as claimed in claim 9, wherein the azido-group is connected directly to the naphthalene ring system.
Claim 12
Dependent Claim
21 words
A method as claimed in claim 9, wherein the azido-group is connected to the naphthalene ring system via a linking group.
Claim 13
Dependent Claim
23 words
The method as claimed in claim 1, wherein the saccharide mixture includes saccharides selected from the group consisting of mono-saccharides, di-saccharides and trisaccharides.
Claim 14
Dependent Claim
13 words
The method as claimed in claim 12, wherein the saccharide is a monosaccharide.
Claim 15
Dependent Claim
13 words
The method as claimed in claim 12, wherein the saccharide is an disaccharide.
Claim 16
Dependent Claim
13 words
The method as claimed in claim 12, wherein the saccharide is a trisaccharide.
Claim 17
Independent Claim
141 words
A method of resolving a mixture of saccharides into distinct groups of closely related or identical saccharides, comprising the steps of: reacting the mixture with a modified form of 1-amino-4-naphthalene sulfonic acid to form modified conjugates, the 1-amino-4-naphthalene sulfonic acid being modified by the addition of a light-sensitive azido-group at the 5, 6, 7 or 8 position of the naphthalene ring system of the 1-amino-4-naphthalene sulfonic acid; subjecting the conjugates to gel electrophoresis in an electrophoresis gel for a sufficient period of time to form separate groups of conjugates in the electrophoresis gel; electro-blotting the separate groups of conjugates in the electrophoresis gel to a membrane surface; and subjecting the conjugates on the membrane surface to light for a sufficient time and of a light frequency which activates the light-sensitive azido-group and creates a binding between the azido-group and the surface.
Claim 18
Independent Claim
141 words
A method of resolving a mixture of saccharides into distinct groups of closely related or identical saccharides, comprising the steps of: reacting the mixture with a modified form of 1-amino-4-naphthalene sulfonic acid to form modified conjugates, the 1-amino-4-naphthalene sulfonic acid being modified by the addition of a light-sensitive azido-group at the 5, 6, 7 or 8 position of the naphthalene ring system of the 1-amino-4-naphthalene sulfonic acid; subjecting the conjugates to gel electrophoresis in an electrophoresis gel for a sufficient period of time to form separate groups of conjugates in the electrophoresis gel; electro-blotting the separate groups of conjugates in the electrophoresis gel to a membrane surface; and subjecting the conjugates on the membrane surface to light for a sufficient time and of a light frequency which activates the light-sensitive azido-group and creates a binding between the azido-group and the surface.
Claim 19
Dependent Claim
15 words
The method as claimed in claim 16, wherein the membrane is a charged nylon membrane.
Claim 20
Dependent Claim
23 words
The method as claimed in claim 17, wherein the saccharide mixture comprises saccharides selected from the group consisting of monosaccharides, disaccharides and trisaccharides.
Claim 21
Dependent Claim
38 words
The method as claimed in claim 18, further comprising: contacting the separate groups of conjugates on the nylon membrane with labeled probes to determine the affinity of the probes to bind to the saccharides in the separate groups.
Claim 22
Dependent Claim
26 words
The method as claimed in claim 19, further comprising: washing away any labeled probes not bound to a saccharide and detecting bound probes by their label.
Claim 23
Independent Claim
147 words
A method for assaying for the affinity of a protein for a saccharide in a mixture of saccharides, comprising the steps of: reacting the mixture of saccharides with a modified form of 1-amino-4-naphthalene sulfonic acid to form conjugates, the 1-amino-4-naphthalene sulfonic acid being modified by the addition of a light-sensitive azido-group; subjecting the conjugates to gel electrophoresis in an electrophoresis gel for a sufficient period of time to form separate groups of conjugates in the electrophoresis gel; electro-blotting the separate groups of conjugates from the gel onto the surface of a membrane; subjecting the conjugates on the membrane to light for a sufficient time and of a light frequency which activates the light-sensitive azido-group which binds to the surface of the membrane; and contacting the membrane having the conjugates thereon with labeled proteins to determine the affinity of the proteins to bind to saccharides on the membrane.
Claim 24
Independent Claim
147 words
A method for assaying for the affinity of a protein for a saccharide in a mixture of saccharides, comprising the steps of: reacting the mixture of saccharides with a modified form of 1-amino-4-naphthalene sulfonic acid to form conjugates, the 1-amino-4-naphthalene sulfonic acid being modified by the addition of a light-sensitive azido-group; subjecting the conjugates to gel electrophoresis in an electrophoresis gel for a sufficient period of time to form separate groups of conjugates in the electrophoresis gel; electro-blotting the separate groups of conjugates from the gel onto the surface of a membrane; subjecting the conjugates on the membrane to light for a sufficient time and of a light frequency which activates the light-sensitive azido-group which binds to the surface of the membrane; and contacting the membrane having the conjugates thereon with labeled proteins to determine the affinity of the proteins to bind to saccharides on the membrane.
Claim 25
Dependent Claim
26 words
The method as claimed in claim 21, further comprising: washing away any labeled proteins not bound to saccharides on the membrane surface and detecting bound proteins.
References Cited (Backward Citations)
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