CA2437381A1 - Improved affinity matrices with enhanced visibility for molecular pull-down and immunoprecipitation applications - Google Patents

Improved affinity matrices with enhanced visibility for molecular pull-down and immunoprecipitation applications Download PDF

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CA2437381A1
CA2437381A1 CA002437381A CA2437381A CA2437381A1 CA 2437381 A1 CA2437381 A1 CA 2437381A1 CA 002437381 A CA002437381 A CA 002437381A CA 2437381 A CA2437381 A CA 2437381A CA 2437381 A1 CA2437381 A1 CA 2437381A1
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affinity matrix
dye
matrix
protein
molecule
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CA2437381C (en
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Edward B. Watson, Iii
Kenneth E. Heuermann
John G. Dapron
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Sigma Aldrich Co LLC
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28004—Sorbent size or size distribution, e.g. particle size
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/328—Polymers on the carrier being further modified
    • B01J20/3282—Crosslinked polymers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B01D15/3823—Affinity chromatography of other types, e.g. avidin, streptavidin, biotin

Abstract

An affinity matrix for use in affinity based molecular pull down and immunoprecipitation procedures. The affinity matrix comprises a polymeric support, a dye attached to a fraction of the polymeric support to enable optical detection of the polymeric support, and an affinity ligand other tha n the dye attached to a fraction of the polymeric support for the capture of a molecule. Also provided is a method for the isolation of a biomolecule from an aqueous solution. The method comprises combining the aqueous solution with a n affinity matrix comprising a polymeric support and separating the affinity matrix from the aqueous solution. A dye is attached to a fraction of the polymeric support which enables optical detection and monitoring of the affinity matrix and, accordingly, reduces the likelihood of the loss of affinity matrix during the separation step. In addition, an affinity ligand other than the dye is also attached to a fraction of the polymeric support f or the capture of the biomolecule.

Claims (97)

1. An affinity matrix for the isolation of a molecule from a aqueous solution, the affinity matrix comprising a polymeric support, a dye attached to a fraction of the polymeric support to enable optical detection of the polymeric support, and an affinity ligand other than the dye attached to a fraction of the polymeric support for the capture of a molecule.
2. The affinity matrix of claim 1 wherein the affinity matrix comprises a particulate polymeric support.
3. The affinity matrix of claim 1 wherein the affinity matrix comprises a particulate polymeric material and the dye and affinity ligands are attached to different fractions of the particulate material.
4. The affinity matrix of claim 3 wherein the dye is attached to about 1 % to about 50% by weight of the particulate polymeric material and the affinity ligand is attached to about 50% to about 99% by weight of the particulate polymeric material.
5. The affinity matrix of claim 3 wherein the dye is attached to about 1 % to about 15% by weight of the particulate polymeric material and the affinity ligand is attached to about 85% to about 99% by weight of the particulate polymeric material.
6. The affinity matrix of claim 3 wherein the dye is attached to about 2% to about 10% by weight of the particulate polymeric material and the affinity ligand is attached to about 90% to about 98% by weight of the particulate polymeric material.
7. The affinity matrix of claim 3 wherein the capacity of the affinity matrix to non-specifically bind protein present in the lysate of a naturally-occurring mammalian cell under physiological salt and pH conditions is less than 100 times the capacity of a reference matrix under the same conditions wherein the reference matrix is substantially identical to the affinity matrix except that the no dye is bound to any of the particulate material of the reference matrix.
8. The affinity matrix of claim 7 wherein the particles have an average size of less than about 1,000 micrometers.
9. The affinity matrix of claim 7 wherein the particles have an average size of less than about 600 micrometers.
10. The affinity matrix of claim 7 wherein the particles have an average size of less than about 400 micrometers.
11. The affinity matrix of claim 7 wherein the particles have an average size of less than about 200 micrometers.
12. The affinity matrix of claim 7 wherein the dye comprises a dye possessing an azo chromophore, a dye possessing an anthroquinone chromophore, or a dye possessing a phthalocyanine chromophore.
13. The affinity matrix of claim 7 wherein the dye comprises a dye possessing a vinyl sulfone, a dye possessing a mono or dichloro triazine, or a dye possessing a monochloro difluoro pyrimidimium.
14. The affinity matrix of claim 7 wherein the dye is selected from a group consisting of 5-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-(phenylazo)-2,7-Naphthalenedisulfonic acid, disodium salt (Procion Red MX-5B), Procion Blue MX-R, Remazol Violet R-4B, Procion Red MX-BRA, 5-(acetylamino)-4-hydroxy-3-[[2-hydroxy-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-2,7-Naphthalenedisulfonic acid, copper complex (Remazol Brilliant Violet 5R), Procion Red MX-GBA, Blue MX-4RD (Navy Blue 21), Blue MX-2G (Cobalt Blue 22), Dharma Fire Red 10, 6-(acetylamino)-4-hydroxy-3-[[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-2-Naphthalenesulfonic acid, disodium salt (Remazol Brilliant Orange 3R, Reactive Orange 16), Remazol Brilliant Red BB
(Reactive Red 21), Procion Turquoise H-A, Remazol Brilliant Scarlet R-3G, Remazol Brilliant Orange R-FN, and Reactive Blue 21.
15. The affinity matrix of claim 3 wherein the capacity of the affinity matrix to non-specifically bind protein present in the lysate of a naturally-occurring mammalian cell under physiological salt and pH conditions is less than 50 times the capacity of a reference matrix under the same conditions wherein the reference matrix is substantially identical to the affinity matrix except that the no dye is bound to any of the particulate material of the reference matrix.
16. The affinity matrix of claim 15 wherein the particulate material has an average size of less than about 1,000 micrometers.
17. The affinity matrix of claim 15 wherein the particulate material has an average size of less than about 600 micrometers.
18. The affinity matrix of claim 15 wherein the particulate material has an average size of less than about 400 micrometers.
19. The affinity matrix of claim 15 wherein the particulate material has an average size of less than about 200 micrometers.
20. The affinity matrix of claim 15 wherein the dye comprises a dye possessing an azo chromophore, a dye possessing an anthroquinone chromophore, or a dye possessing a phthalocyanine chromophore.
21. The affinity matrix of claim 15 wherein the dye comprises a dye possessing a vinyl sulfone, a dye possessing a mono or dichloro triazine, or a dye possessing a monochloro difluoro pyrimidimium.
22. The affinity matrix of claim 15 wherein the dye is selected from a group consisting of 6-(acetylamino)-4-hydroxy-3-[[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-2-Naphthalenesulfonic acid, disodium salt (Remazol Brilliant Orange 3R, Reactive Orange 16), Remazol Brilliant Red BB
(Reactive Red 21), Procion Turquoise H-A, Remazol Brilliant Scarlet R-3G, Remazol Brilliant Orange R-FN, Remazol Violet R-4B, 5-(acetylamino)-4-hydroxy-3-[[2-hydroxy-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-2,7-Naphthalenedisulfonic acid, copper complex (Remazol Brilliant Violet 5R), and Reactive Blue 21.
23. The affinity matrix of claim 3 wherein the capacity of the affinity matrix to non-specifically bind protein present in the lysate of a naturally-occurring mammalian cell under physiological salt and pH conditions is less than 10 times the capacity of a reference matrix under the same conditions wherein the reference matrix is substantially identical to the affinity matrix except that the no dye is bound to any of the particulate material of the reference matrix.
24. The affinity matrix of claim 23 wherein the particulate material has an average size of less than about 1,000 micrometers.
25. The affinity matrix of claim 23 wherein the particulate material has an average size of less than about 600 micrometers.
26. The affinity matrix of claim 23 wherein the particulate material has an average size of less than about 400 micrometers.
27. The affinity matrix of claim 23 wherein the particulate material has an average size of less than about 200 micrometers.
28. The affinity matrix of claim 23 wherein the dye comprises a dye possessing an azo chromophore, a dye possessing an anthroquinone chromophore, or a dye possessing a phthalocyanine chromophore.
29. The affinity matrix of claim 23 wherein the dye comprises a dye possessing a vinyl sulfone, a dye possessing a mono or dichloro triazine, or a dye possessing a monochloro difluoro pyrimidimium.
30. The affinity matrix of claim 23 wherein the dye is selected from a group consisting of 6-(acetylamino)-4-hydroxy-3-[[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-2-Naphthalenesulfonic acid, disodium salt (Remazol Brilliant Orange 3R, Reactive Orange 16), Remazol Brilliant Red BB
(Reactive Red 21), Remazol Brilliant Scarlet R-3G, Remazol Brilliant Orange R-FN, and Reactive Blue 21.
31. The affinity matrix of claim 3 wherein the capacity of the affinity matrix to non-specifically bind protein present in the lysate of a naturally-occurring mammalian cell under physiological salt and pH conditions is less than 3 times the capacity of a reference matrix under the same conditions wherein the reference matrix is substantially identical to the affinity matrix except that the no dye is bound to any of the particulate material of the reference matrix.
32. The affinity matrix of claim 31 wherein the particulate material has an average size of less than about 1,000 micrometers.
33. The affinity matrix of claim 31 wherein the particulate material has an average size of less than about 600 micrometers.
34. The affinity matrix of claim 31 wherein the particulate material has an average size of less than about 400 micrometers.
35. The affinity matrix of claim 31 wherein the particulate material has an average size of less than about 200 micrometers.
36. The affinity matrix of claim 31 wherein the dye comprises a dye possessing an azo chromophore, a dye possessing an anthroquinone chromophore, or a dye possessing a phthalocyanine chromophore.
37. The affinity matrix of claim 31 wherein the dye comprises a dye possessing a vinyl sulfone, a dye possessing a mono or dichloro triazine, or a dye possessing a monochloro difluoro pyrimidimium.
38. The affinity matrix of claim 31 wherein the dye comprises 6-(acetylamino)-4-hydroxy-3-[[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-2-Naphthalenesulfonic acid, disodium salt (Remazol Brilliant Orange 3R, Reactive Orange 16) or Remazol Brilliant Red BB (Reactive Red 21).
39. The affinity matrix of claim 31 wherein the dye comprises Remazol Brilliant Red BB (Reactive Red 21).
40. The affinity matrix of claim 3 wherein the particulate material has an average size of less than about 1,000 micrometers.
41. The affinity matrix of claim 3 wherein the particulate material has an average size of less than about 600 micrometers.
42. The affinity matrix of claim 3 wherein the particulate material has an average size of less than about 400 micrometers.
43. The affinity matrix of claim 3 wherein the particulate material has an average size of less than about 200 micrometers.
44. The affinity matrix of claim 1 wherein the affinity matrix is modified by gelatin absorption and cross-linking.
45. The affinity matrix of claim 44 wherein the dye comprises a dye possessing an azo chromophore, an anthroquinone chromophore, or a phthalocyanine chromophore.
46. The affinity matrix of claim 44 wherein the dye comprises a dye possessing a vinyl sulfone, a dye possessing a mono or dichloro triazine, or a dye possessing a monochloro difluoro pyrimidimium.
47. The affinity matrix of claim 44 wherein the dye comprises Reactive Red 120 or Reactive Brown 10.
48. The affinity matrix of claim 44 wherein the dye comprises fluorescein, a coumarin derivative, a pyridyloxazole derivative, or a lanthanide metal chelate.
49. The affinity matrix of claim 44 wherein the dye comprises napthalene, pyrene, rhodamine, fluorescein isothiocyanate, a terbium complex, a europium complex, or a ruthenium complex.
50. The affinity matrix of claim 1 wherein the polymeric support comprises natural agarose, cross-linked agarose, cross-linked dextran, or polyacrylamide
51. The affinity matrix of claim 2 wherein the particulate polymeric support comprises natural agarose particles, cross-linked agarose particles, cross-linked dextran particles, or polyacrylamide particles.
52. The affinity matrix of claim 50 wherein the cross-linked agarose comprises particles about 20 to about 300 micrometers in diameter.
53. The affinity matrix of claim 50 wherein the cross-linked agarose comprises particles about 30 to about 250 micrometers in diameter.
54. The affinity matrix of claim 50 wherein the cross-linked agarose comprises particles about 40 to about 165 micrometers in diameter.
55. The affinity matrix of claim 50 wherein the natural agarose comprises particles about 30 to about 250 micrometers in diameter.
56. The affinity matrix of claim 50 wherein the cross-linked dextran comprises particles about 20 to about 400 micrometers in diameter.
57. The affinity matrix of claim 50 wherein the polyacrylamide comprises particles about 30 to about 150 micrometers in diameter.
58. The affinity matrix of claim 1 wherein the dye is directly covalently attached to the polymer.
59. The affinity matrix of claim 1 wherein the amount of dye that is attached to the polymeric support comprises between about 0.5 micromoles of dye/cm3 of polymeric support to about 20 micromoles of dye/cm3 of polymeric support.
60. The affinity matrix of claim 1 wherein the amount of dye that is attached to the polymeric support comprises between about 1 micromole of dye/cm3 of polymeric support to about 6 micromoles of dye/cm3 of polymeric support.
61. The affinity matrix of claim 1 wherein the amount of dye that is attached to the polymeric support comprises between about 2 micromoles of dye/cm3 of polymeric support to about 4 micromoles of dye/cm3 of polymeric support.
62. The affinity matrix of claim 1 wherein the polymer comprises cross-linked agarose particles ranging from about 30 to about 250 micrometers in diameter, said cross-linked agarose particles possessing between about 1 micromole of said dye/cm3 of cross-linked agarose to about 6 micromoles of said dye /cm3 of cross-linked agarose.
63. The affinity matrix of claim 1 wherein the polymer comprises cross-linked agarose particles ranging from about 40 to about 165 micrometers in diameter, said cross-linked agarose particles possessing between about 2 micromole of said dye/cm3 of cross-linked agarose to about 4 micromoles of said dye /cm3 of cross-linked agarose.
64. The affinity matrix of claim 1 wherein the affinity ligands are selected from the group consisting of streptavidin, monomeric streptavidin, a molecule with the binding properties of streptavidin, avidin, monomeric avidin, a molecule with the binding properties of avidin, streptactin, monomeric streptactin, a molecule with the binding properties of streptactin, extravidin, monomeric extravidin, a molecule with the binding properties of extravidin, neutravidin, monomeric neutravidin, a molecule with the binding properties of neutravidin, protein A, a molecule with the binding properties of protein A, protein L, a molecule with the binding properties of protein L, protein G, a molecule with the binding properties of protein G, protein A/G, a molecule with the binding properties of protein A/G, protein L/A, a molecule with the binding properties of protein L/A, calmodulin, a molecule with the binding properties of calmodulin, biotin, a molecule with the binding properties of biotin, metal chelates, glutathione, and antibodies.
65. The affinity matrix of claim 1 wherein the affinity matrix is collectable from the solution by centrifugation of less than 700 x g.
66. The affinity matrix of claim 1 comprising a mixture of a non-colored affinity matrix and a colored polymeric support, the colored polymeric support comprising a polymer and a dye attached to the polymer.
67. The affinity matrix of claim 66 wherein the colored polymeric support comprises between about 1% by volume to about 95% by volume of the affinity matrix.
68. The affinity matrix of claim 66 wherein the colored polymeric support comprises between about 2% by volume to about 50% by volume of the affinity matrix.
69. The affinity matrix of claim 66 wherein the colored polymeric support comprises between about 2% by volume to about 10% by volume of the affinity matrix.
70. The affinity matrix of claim 66 wherein the non-colored affinity matrix comprises an antibody binding matrix.
71. The affinity matrix of claim 70 wherein the antibody binding matrix is selected from the group consisting of protein A agarose, agarose modified with a molecule with the binding properties of protein A, protein G agarose, agarose modified with a molecule with the binding properties of protein G, protein L
agarose, agarose modified with a molecule with the binding properties of protein L, protein A/G agarose, agarose modified with a molecule with the binding properties of protein A/G, protein L/A agarose, and agarose modified with a molecule with the binding properties of protein L/A.
72. The affinity matrix of claim 66 wherein the non-colored affinity matrix comprises a matrix with attached antibodies.
73. The affinity matrix of claim 72 wherein the matrix with attached antibodies comprises a matrix with antibodies to particular proteins.
74. The affinity matrix of claim 72 wherein the matrix with attached antibodies comprises a matrix with antibodies to specific peptide tags on fusion proteins.
75. The affinity matrix of claim 72 wherein the matrix with attached antibodies comprises a matrix with antibodies to epitopes within proteins.
76. The affinity matrix of claim 72 wherein the antibodies are selected from the group consisting of antibodies having specificity for at least a portion of the amino acid sequence DYKDDDDK anti-FLAG.TM. ("DYKDDDDK"), anti-polyhistidine, anti-glutathione-S-transferase, anti-myc-tag, anti-Avi-tag, anti-HA, anti-Green fluorescent protein, anti-beta galactosidase, anti-thioredoxin, anti-maltose binding protein, anti-cellulose binding domain, anti-VSV glycoprotein, and anti-luciferase.
77. The affinity matrix of claim 66 wherein the non-colored affinity matrix comprises a matrix with attached affinity ligands.
78. The affinity matrix of claim 77 wherein the affinity ligands are selected from the group consisting of streptavidin, monomeric streptavidin, a molecule with the binding properties of streptavidin, avidin, monomeric avidin, a molecule with the binding properties of avidin, streptactin, monomeric streptactin, a molecule with the binding properties of streptactin, extravidin, monomeric extravidin, a molecule with the binding properties of extravidin, neutravidin, monomeric neutravidin, a molecule with the binding properties of neutravidin, protein A, a molecule with the binding properties of protein A, protein L, a molecule with the binding properties of protein L, protein G, a molecule with the binding properties of protein G, protein A/G, a molecule with the binding properties of protein A/G, protein L/A, a molecule with the binding properties of protein L/A, calmodulin, a molecule with the binding properties of calmodulin, biotin, a molecule with the binding properties of biotin, metal chelates, and glutathione.
79. A method for the isolation of a biomolecule from a sample aqueous solution, the method comprising combining the aqueous solution with an affinity matrix comprising a polymeric support, a dye attached to a fraction of the polymeric support to enable optical detection of the affinity matrix, and an affinity ligand other than the dye attached to a fraction of the polymeric support for the capture of the biomolecule, and separating the affinity matrix from the sample aqueous solution.
80. The method of claim 79 wherein the affinity matrix is optically monitored during the separation step to avoid the loss of affinity matrix.
81. The method of claim 79 wherein the separation step is carried out manually and the affinity matrix is visually monitored during the separation to avoid the loss of affinity matrix.
82. The method of claim 79 wherein the affinity ligand is selected from the group consisting of streptavidin, monomeric streptavidin, a molecule with the binding properties of streptavidin, avidin, monomeric avidin, a molecule with the binding properties of avidin, streptactin, monomeric streptactin, a molecule with the binding properties of streptactin, extravidin, monomeric extravidin, a molecule with the binding properties of extravidin, neutravidin, monomeric neutravidin, a molecule with the binding properties of neutravidin, protein A, a molecule with the binding properties of protein A, protein L, a molecule with the binding properties of protein L, protein G, a molecule with the binding properties of protein G, protein A/G, a molecule with the binding properties of protein A/G, protein L/A, a molecule with the binding properties of protein L/A, calmodulin, a molecule with the binding properties of calmodulin, biotin, and a molecule with the binding properties of biotin, metal chelates, glutathione, and antibodies.
83. The method of claim 79 wherein the aqueous sample solution containing the biomolecule is added to the affinity matrix.
84. The method of claim 79 wherein the method is an immunoprecipitation.
85. The method of claim 79 wherein the biomolecule comprises a naturally occurring biomolecule, a synthetic biomolecule, a modified naturally occurring biomolecule, or a modified synthetic biomolecule.
86. The method of claim 85 wherein the biomolecule is selected from the group consisting of (i) peptides, polypeptides, individual proteins, glycoproteins, enzymes, nucleotides, polynucleotides, nucleic acid polymers, carbohydrates, lipids, and (ii) complexes containing a biomolecule of group (i).
87. The method of claim 85 wherein the biomolecule comprises a protein.
88. The method of claim 79 wherein the aqueous sample solution is selected from the group consisting of a lysate, lysate fractions, a buffered solution containing unpurified biomolecules, and a buffered solution containing purified biomolecules.
89. The method of claim 79 wherein the aqueous sample solution comprises a lysate.
90. The method of claim 79 wherein the process additionally comprises the step of washing the affinity matrix with an aqueous wash solution after it has been separated from the sample solution, separating the affinity matrix from the aqueous wash solution, and optically monitoring the affinity matrix as it is separated from the aqueous wash solution.
91. The method of claim 90 wherein the aqueous wash solution comprises a lysis buffer.
92. The method of claim 79 further comprising releasing the biomolecules which are bound to the affinity matrix.
93. The method of claim 90 further comprising releasing the biomolecules which are bound to the affinity matrix.
94. The method of claim 92 further comprising subjecting the biomolecules to a method of analysis.
95. The method of claim 93 further comprising subjecting the biomolecules to a method of analysis.
96. The method of claim 95 wherein the method of analysis comprises SDS-PAGE.
97. The method of claim 95 wherein the method of analysis comprises an enzymatic assay.
CA2437381A 2001-02-01 2002-01-31 Improved affinity matrices with enhanced visibility for molecular pull-down and immunoprecipitation applications Expired - Lifetime CA2437381C (en)

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Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2545526T3 (en) * 2001-02-01 2015-09-11 Sigma-Aldrich Co. Llc Enhanced affinity matrices with enhanced visibility for molecular drag and immunoprecipitation applications
US6825032B2 (en) * 2001-05-14 2004-11-30 Sigma-Aldrich Co. High capacity assay platforms
WO2004081570A1 (en) * 2003-03-13 2004-09-23 Chengdu Kuachang Medical Industrial Limited A chip matrix, a chip comprising the matrix and their preparation and application
WO2006071970A2 (en) * 2004-12-23 2006-07-06 Georgetown University Methods for the detection of analytes in a sample
US7479324B2 (en) * 2005-11-08 2009-01-20 Saint-Gobain Ceramics & Plastics, Inc. Pigments comprising alumina hydrate and a dye, and polymer composites formed thereof
WO2008028218A1 (en) * 2006-09-05 2008-03-13 Innovative Purification Technologies Pty Ltd Affinity separation methods and systems
JP4954004B2 (en) * 2007-08-09 2012-06-13 株式会社バイオダイナミクス研究所 Novel nucleic acid derivative into which an arbitrary compound is introduced, and production method thereof
PL2427482T3 (en) * 2009-05-07 2020-08-24 Chreto Aps Method for purification of target polypeptides
CN102062780A (en) * 2010-11-23 2011-05-18 北京正旦国际科技有限责任公司 Polypeptide immunoassay kit and detection method thereof
EP2568289A3 (en) 2011-09-12 2013-04-03 International AIDS Vaccine Initiative Immunoselection of recombinant vesicular stomatitis virus expressing hiv-1 proteins by broadly neutralizing antibodies
US9402894B2 (en) 2011-10-27 2016-08-02 International Aids Vaccine Initiative Viral particles derived from an enveloped virus
US9347065B2 (en) 2012-03-29 2016-05-24 International Aids Vaccine Initiative Methods to improve vector expression and genetic stability
JP7009822B2 (en) 2016-08-09 2022-02-10 東ソー株式会社 Detection method using fibrous substance
CN107267475A (en) * 2017-08-11 2017-10-20 浙江福斯特新材料研究院有限公司 A kind of method that metal chelate affinity chromatography purifies thioredoxin

Family Cites Families (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4166105A (en) * 1973-07-30 1979-08-28 Block Engineering, Inc. Dye tagged reagent
DE2363854C3 (en) * 1973-12-21 1980-01-03 Behringwerke Ag, 3550 Marburg Substrate for proteinase determination and its production
US4108972A (en) * 1974-03-15 1978-08-22 Dreyer William J Immunological reagent employing radioactive and other tracers
US4016133A (en) 1974-03-18 1977-04-05 Sinloihi Co. Fluorescent colored resin particles and process for preparation thereof
JPS635133B2 (en) 1978-01-24 1988-02-02 Paburitsuku Herusu Raboratarii Saauisu Boodo
NL8000173A (en) 1980-01-11 1981-08-03 Akzo Nv USE OF WATER-DISPERSIBLE HYDROPHOBIC DYES AS LABELS IN IMMUNOCHEMICAL TESTS.
DE3271828D1 (en) 1981-04-27 1986-07-31 Health Lab Service Board High pressure liquid affinity chromatography
US4434150A (en) * 1981-10-19 1984-02-28 Ortho Diagnostic Systems, Inc. Immunological reagents employing polymeric backbone possessing reactive functional groups
EP0126772A1 (en) 1982-12-03 1984-12-05 E.I. Du Pont De Nemours And Company Chromogenic support immunoassay
US4605630A (en) 1983-07-27 1986-08-12 Cooper Lipotech Inc. Large-liposome agglutination reagent and method
JPS61124392A (en) 1984-11-22 1986-06-12 Asahi Chem Ind Co Ltd Purification of physiologically active substance produced by gene recombinant
EP0190606B1 (en) 1985-02-04 1990-05-09 F. Hoffmann-La Roche Ag Sorbent for the purification of proteins
US4707523A (en) 1985-10-11 1987-11-17 California Institute Of Technology Magnetic particles
US5395688A (en) 1987-10-26 1995-03-07 Baxter Diagnostics Inc. Magnetically responsive fluorescent polymer particles
US5597485A (en) 1988-05-13 1997-01-28 Vilmax S.A. Process for separating proteins
DE3921498A1 (en) 1988-09-28 1990-03-29 Bayer Ag POLYMER-TIED DYES, METHOD FOR THE PRODUCTION AND USE THEREOF
ATE175681T1 (en) 1988-10-17 1999-01-15 Hemasure Inc METHOD FOR THE COVALENT SURFACE MODIFICATION OF HYDROPHOBIC POLYMERS AND PRODUCTS THEREOF
JPH04503109A (en) 1989-01-30 1992-06-04 エピトープ，インコーポレイティド Avidin-biotin assisted immunoassay
US5491097A (en) * 1989-06-15 1996-02-13 Biocircuits Corporation Analyte detection with multilayered bioelectronic conductivity sensors
GB8915654D0 (en) 1989-07-07 1989-08-23 Unilever Plc Detectable particles
US5232830A (en) 1990-05-11 1993-08-03 Microprobe Corporation Intrinsic fluorescent quenching methods
US5135627A (en) * 1990-10-15 1992-08-04 Soane Technologies, Inc. Mosaic microcolumns, slabs, and separation media for electrophoresis and chromatography
JPH04166767A (en) 1990-10-30 1992-06-12 Japan Synthetic Rubber Co Ltd Gelatin coated particle
US5316680A (en) * 1992-10-21 1994-05-31 Cornell Research Foundation, Inc. Multimodal chromatographic separation media and process for using same
SE9300090D0 (en) 1993-01-14 1993-01-14 Bo Gustav Mattiasson AFFINITY CLEANING WITH COMPLEX BOND LIGAND
US5914241A (en) 1993-01-19 1999-06-22 Biosite Diagnostics, Inc. Assays and kits for detecting analytes in the presence of cross-reacting substances
JP2871435B2 (en) 1993-04-22 1999-03-17 イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー Method for producing a hydrophilic polymer coated perfluorocarbon polymer based matrix
CA2106612C (en) * 1993-09-21 2001-02-06 Diana Pliura Displacement chromatography process
US5643721A (en) * 1994-02-09 1997-07-01 Abbott Laboratories Bioreagent immobilization medium
US5728457A (en) * 1994-09-30 1998-03-17 Cornell Research Foundation, Inc. Porous polymeric material with gradients
US5522994A (en) * 1995-02-01 1996-06-04 Cornell Research Foundation, Inc. Single column chromatographic determination of small molecules in mixtures with large molecules
US6017496A (en) 1995-06-07 2000-01-25 Irori Matrices with memories and uses thereof
US5587323A (en) * 1995-05-02 1996-12-24 United States Drug Testing Laboratories, Inc. Meconium assay procedure
DE69620898T2 (en) 1995-09-01 2002-11-07 Univ Washington Seattle INTERACTIVE MOLECULAR CONJUGATES
ATE263254T1 (en) 1995-10-02 2004-04-15 Emd Biosciences Inc METHOD FOR THE PRECIPITATION OF NUCLEIC ACIDS WITH A VISIBLE SUPPORT
EP1386660B1 (en) 1996-08-30 2009-09-02 Upfront Chromatography A/S Isolation of immunoglobulins
US6555661B1 (en) 1997-08-25 2003-04-29 Anthony R. Torres Simple, environmentally benign, method for purifying protein A
GB9816514D0 (en) 1998-07-29 1998-09-30 Smithkline Beecham Plc Novel method
EP1103812B1 (en) * 1998-08-07 2015-11-18 Sekisui Chemical Co., Ltd. Method for determining hemoglobins
JP4863548B2 (en) 1998-09-08 2012-01-25 ヘンリー フォード ヘルス システム Method of detecting, preventing and treating renal injury by modulation, regulation and inhibition of connective tissue growth factor
US6242185B1 (en) * 1999-04-01 2001-06-05 Incyte Genomics, Inc. Purified nucleic acid encoding transcription factor regulatory protein
ES2545526T3 (en) * 2001-02-01 2015-09-11 Sigma-Aldrich Co. Llc Enhanced affinity matrices with enhanced visibility for molecular drag and immunoprecipitation applications

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