US4512896A
Expired / Lapsed
US
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Transfer of macromolecules from a chromatographic substrate to an immobilizing matrix
Assignee: Yale University
•
Filing: 1983-02-07
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Granted: 1985-04-23
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Expires: 2003-02-07
Abstract
Abstract The method of transfer of macromolecules such as nucleic acid and proteins from a chromatographic substrate to an immobilizing matrix uses as the immobilizing matrix, a charge modified microporous membrane comprising an organic microporous membrane having a charge modifying amount of a cationic charge modifying agent bonded to substantially all of the wetted surfaces of said membrane. The charge modified microporous membrane can also be a reinforced microporous membrane, preferably a porous reinforcing web impregnated with a polymeric microporous membrane. A nucleic acid or protein blotting product comprising a chromatographic matrix having the charge modified microporous membrane on a surface thereof is also provided.
Inventors
- 👤 Jonathan M. Gershoni
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 02:20
CPC Classifications
B; B01; B01D; B01D67/00; B01D67/0081; B01D67/0093; B01D67/00931; B01D57/00; B01D57/02; B01D71/00; B01D71/06; B01D71/56; B01J; B01J20/00; B01J20/281; B01J20/286; B01J20/30; B01J20/32; B01J20/3202; B01J20/3206; B01J20/3208; B01J20/3212; B01J20/3231; B01J20/3242; B01J20/3268; B01J20/3272; B01J20/328; B01J20/3282; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/543; G01N33/544; G01N33/545; B01D2323/00; B01D2323/30
Claims Analysis
Total Claims: 57
Claim 1
Independent Claim
62 words
A product comprising a chromatographic substrate having on a surface thereof as an immobilizing matrix a hydrophilic charge modified microporous membrane comprising an organic microporous membrane having a charge modifying amount of a cationic charge modifying agent bonded to substantially all of the surfaces of said membrane wetted by a fluid passing through said membrane or in which said membrane is immersed.
Claim 2
Dependent Claim
62 words
A product comprising a chromatographic substrate having on a surface thereof as an immobilizing matrix a hydrophilic charge modified microporous membrane comprising an organic microporous membrane having a charge modifying amount of a cationic charge modifying agent bonded to substantially all of the surfaces of said membrane wetted by a fluid passing through said membrane or in which said membrane is immersed.
Claim 3
Dependent Claim
12 words
The product of claim 1, wherein the organic microporous membrane is hydrophilic.
Claim 4
Dependent Claim
16 words
The product of claim 2, wherein the hydrophilic organic polymeric microporous membrane is a cellulose ester.
Claim 5
Dependent Claim
15 words
The product of claim 2, wherein the hydrophilic organic polymeric microporous membrane is a polyamide.
Claim 6
Dependent Claim
51 words
The product of claim 2, wherein the charge modifying agent comprises a water-soluble organic polymer having a molecular weight greater than about 1,000, wherein each monomer thereof has least one epoxide group capable of bonding to the surface of the membrane and at least one tertiary amine or quaternary ammonium group.
Claim 7
Dependent Claim
15 words
The product of claim 5, wherein said water-soluble organic polymer is a polyamido-polyamine epichlorohydrin resin.
Claim 8
Dependent Claim
18 words
The macromolecule blotting product of claim 6 is a polymer having repeating monomer units of the formula: ##STR7##
Claim 9
Dependent Claim
11 words
The product of claim 7, wherein said membrane is polyhexamethylene adipamide.
Claim 10
Dependent Claim
12 words
The product of claim 8, wherein said substrate is an electrophoresis gel.
Claim 11
Dependent Claim
10 words
The product of claim 9, wherein said gel comprises polyacrylamide.
Claim 12
Dependent Claim
65 words
The product of claim 5, wherein a portion of the epoxy groups of the organic polymer charge modifying agent are bonded to a secondary charge modifying agent selected from the group consisting of: (i) aliphatic amines having at least one primary amino or at least two secondary amino groups; and (ii) aliphatic amines having at least one secondary amino and a carboxyl or hydroxyl substituent.
Claim 13
Dependent Claim
13 words
The product of claim 11, wherein said secondary charge modifying agent is tetraethylenepentamine.
Claim 14
Dependent Claim
12 words
The product of claim 12, wherein said substrate is an electrophoresis gel.
Claim 15
Dependent Claim
77 words
The product of claim 2, wherein said charge modifying agent is selected from the group consisting of: (i) aliphatic amines having at least one primary amino or at least two secondary amino groups; and (ii) aliphatic amines having at least one secondary amino and a carboxyl or hydroxyl substituent, and wherein said charge modifying agent is bonded to the microporous membrane structure through an aliphatic polyepoxide crosslinking agent having a molecular weight of less than about 500.
Claim 16
Dependent Claim
12 words
The product of claim 14, wherein the polyepoxide is 1,4-butanediol diglycidal ether.
Claim 17
Dependent Claim
12 words
The product of claim 15, wherein said charge modifying agent is tetraethylenepentamine.
Claim 18
Dependent Claim
12 words
The product of claim 16, wherein said substrate is an electrophoresis gel.
Claim 19
Dependent Claim
20 words
The product of claim 4, wherein the membrane has a pore size of from about 0.05 to about 1.2 microns.
Claim 20
Dependent Claim
71 words
In a method for the transfer of macromolecule from a chromatographic substrate to an immobilizing matrix, the improvement which comprises employing as said matrix, a hydrophilic charge modified microporous membrane comprising an organic microporous membrane having a charge modifying amount of a cationic charge modifying agent bonded to substantially all of the surfaces of said membrane wetted by a fluid passing through said membrane or in which said membrane is immersed.
Claim 21
Dependent Claim
71 words
In a method for the transfer of macromolecule from a chromatographic substrate to an immobilizing matrix, the improvement which comprises employing as said matrix, a hydrophilic charge modified microporous membrane comprising an organic microporous membrane having a charge modifying amount of a cationic charge modifying agent bonded to substantially all of the surfaces of said membrane wetted by a fluid passing through said membrane or in which said membrane is immersed.
Claim 22
Dependent Claim
12 words
The method of claim 19, wherein said organic microporous membrane is hydrophilic.
Claim 23
Dependent Claim
16 words
The method of claim 20, wherein the hydrophilic organic polymeric microporous membrane is a cellulose ester.
Claim 24
Dependent Claim
15 words
The method of claim 20, wherein the hydrophilic organic polymeric microporous membrane is a polyamide.
Claim 25
Dependent Claim
51 words
The method of claim 22, wherein the charge modifying agent comprises a water-soluble organic polymer having a molecular weight greater than about 1,000, wherein each monomer thereof has least one epoxide group capable of bonding to the surface of the membrane and at least one tertiary amine or quaternary ammonium group.
Claim 26
Dependent Claim
15 words
The method of claim 23, wherein said water-soluble organic polymer is a polyamido-polyamine epichlorohydrin resin.
Claim 27
Dependent Claim
16 words
The method of claim 24, wherein said polymer has repeating monomer units of the formula: ##STR8##
Claim 28
Dependent Claim
12 words
The method of claim 25, wherein said membrane is a polyhexamethylene adipamide.
Claim 29
Dependent Claim
12 words
The method of claim 25, wherein said substrate is an electrophoresis gel.
Claim 30
Dependent Claim
10 words
The method of claim 27, wherein said gel comprises polyacrylamide.
Claim 31
Dependent Claim
65 words
The method of claim 23, wherein a portion of the epoxy groups of the organic polymer charge modifying agent are bonded to a secondary charge modifying agent selected from the group consisting of: (i) aliphatic amines having at least one primary amino or at least two secondary amino groups; and (ii) aliphatic amines having at least one secondary amino and a carboxyl or hydroxyl substituent.
Claim 32
Dependent Claim
13 words
The method of claim 29, wherein said secondary charge modifying agent is tetraethylenepentamine.
Claim 33
Dependent Claim
12 words
The method of claim 30, wherein said substrate is an electrophoresis gel.
Claim 34
Dependent Claim
77 words
The method of claim 20, wherein said charge modifying agent is selected from the group consisting of: (i) aliphatic amines having at least one primary amino or at least two secondary amino groups; and (ii) aliphatic amines having at least one secondary amino and a carboxyl or hydroxyl substituent, and wherein said charge modifying agent is bonded to the microporous membrane structure through an aliphatic polyepoxide crosslinking agent having a molecular weight of less than about 500.
Claim 35
Dependent Claim
12 words
The method of claim 32, wherein the polyepoxide is 1,4-butanediol diglycidal ether.
Claim 36
Dependent Claim
12 words
The method of claim 33, wherein said charge modifying agent is tetraethylenepentamine.
Claim 37
Dependent Claim
12 words
The method of claim 20, wherein said substrate is an electrophoresis gel.
Claim 38
Dependent Claim
20 words
The method of claim 22, wherein the membrane has a pore size of from about 0.05 to about 1.2 microns.
Claim 39
Dependent Claim
14 words
The method of claim 20, wherein the transferred protein is incubated with a ligand.
Claim 40
Dependent Claim
24 words
The method of claim 37, wherein the charge modified microporous membrane is quenched after transfer of the protein and before incubation with the ligand.
Claim 41
Dependent Claim
23 words
The method of claim 38, wherein the quenching is effected by incubating the membrane with bovine serum albumin or hemoglobin at elevated temperature.
Claim 42
Dependent Claim
11 words
The method of claim 22, wherein said transfer is by electroelution.
Claim 43
Dependent Claim
71 words
In a solid phase assay system wherein a macromolecule is immobilized in the solid phase, the improvement which comprises employing as said solid phase, a hydrophilic charge modified microporous membrane comprising an organic microporous membrane having a charge modifying amount of a cationic, charge modifying agent bonded to substantially all of the surfaces of said membrane wetted by a fluid passing through said membranes or in which said membrane is immersed.
Claim 44
Dependent Claim
71 words
In a solid phase assay system wherein a macromolecule is immobilized in the solid phase, the improvement which comprises employing as said solid phase, a hydrophilic charge modified microporous membrane comprising an organic microporous membrane having a charge modifying amount of a cationic, charge modifying agent bonded to substantially all of the surfaces of said membrane wetted by a fluid passing through said membranes or in which said membrane is immersed.
Claim 45
Dependent Claim
12 words
The system of claim 41, wherein said organic microporous membrane is hydrophilic.
Claim 46
Dependent Claim
16 words
The system of claim 42, wherein the hydrophilic organic polymeric microporous membrane is a cellulose ester.
Claim 47
Dependent Claim
15 words
The system of claim 42, wherein the hydrophilic organic polymeric microporous membrane is a polyamide.
Claim 48
Dependent Claim
52 words
The system of claim 43, wherein the charge modifying agent comprises a water-soluble organic polymer having a molecular weight greater than about 1,000, wherein each monomer thereof has at least one epoxide group capable of bonding to the surface of the membranes and at least one tertiary amine or quaternary ammonium group.
Claim 49
Dependent Claim
15 words
The system of claim 44, wherein said water-soluble organic polymer is a polyamido-polyamine epichlorohydrin resin.
Claim 50
Dependent Claim
16 words
The system of claim 46, wherein said polymer has repeating monomer units of the formula: ##STR9##
Claim 51
Dependent Claim
12 words
The system of claim 47, wherein said membrane is a polyhexamethylene adipamide.
Claim 52
Dependent Claim
65 words
The system of claim 45, wherein a portion of the epoxy groups of the organic polymer charge modifying agent are bonded to a secondary charge modifying agent selected from the group consisting of: (i) aliphatic amines having at least one primary amino or at least two secondary amino groups; and (ii) aliphatic amines having at least one secondary amino and a carboxyl or hydroxyl substituent.
Claim 53
Dependent Claim
13 words
The system of claim 49, wherein said secondary charge modifying agent is tetraethylenepentamine.
Claim 54
Dependent Claim
77 words
The system of claim 41, wherein said charge modifying agent is selected from the group consisting of: (i) aliphatic amines having at least one primary amino or at least two secondary amino groups; and (ii) aliphatic amines having at least one secondary amino and a carboxyl or hydroxyl substituent, and wherein said charge modifying agent is bonded to the microporous membrane structure through an aliphatic polyepoxide crosslinking agent having a molecular weight of less than about 500.
Claim 55
Dependent Claim
12 words
The system of claim 51, wherein the polyepoxide is 1,4-butanediol diglycidal ether.
Claim 56
Dependent Claim
12 words
The system of claim 52, wherein said charge modifying agent is tetraethylenepentamine.
Claim 57
Dependent Claim
15 words
The system of claim 42, wherein the charge modified miciroporous membrane is a quenched membrane.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1988-04-22 | 1988-10-22 | 1989-04-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1992-04-22 | 1992-10-22 | 1993-04-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1996-04-22 | 1996-10-22 | 1997-04-23 | $7700.00 | Window Closed / Lapsed |