Claims
- 1. A method of detecting biomolecules in a sample comprising:
providing a stack of at least two layered membranes; applying the sample to the stack under conditions that permit movement of the biomolecules through multiple layered membranes of the stack, and allow direct capture of at least a portion of the biomolecules on the membranes; and detecting the biomolecules on one or more of the multiple membranes.
- 2. The method according to claim 1 wherein the stack comprises a plurality of porous substrates each having a thickness of less than 30 microns.
- 3. The method according to claim 2 wherein one or more of the substrates comprise a material for increasing the affinity of the membrane to the biomolecules.
- 4. The method of claim 3, wherein the material is coated on one or more of the membranes.
- 5. The method of claim 2 wherein the porous substrates comprise a material selected from the group consisting of polycarbonate, cellulose acetate, and mixtures thereof.
- 6. The method of claim 5, wherein the porous substrate comprises a polycarbonate substrate.
- 7. The method of claim 5, wherein the material for increasing affinity is selected from the group consisting of nitrocellulose, poly-L-lysine, and mixtures thereof.
- 8. The method of claim 5, wherein the material for increasing affinity is a biomolecule-specific ligand.
- 9. The method of claim 5, wherein the porous substrate comprises a polycarbonate substrate and the material for increasing affinity comprises nitrocellulose.
- 10. The method according to claim 1 wherein the sample is a tissue section.
- 11. The method of claim 1, wherein detecting the biomolecules comprises separating one or more of the membranes from the stack, and detecting the biomolecules on the one or more of the separated membranes.
- 12. The method of claim 1, wherein the conditions that permit movement of the biomolecules through the multiple membranes comprises passing a transfer liquid through the layered membranes.
- 13. The method of claim 1, wherein the conditions that permit movement of the biomolecules through one or more of the membranes comprises providing a wick that encourages movement of the biomolecules through the stack of layered membranes in a desired direction of movement.
- 14. The method of claim 1, wherein the stack of layered membranes comprises 5 or more membranes.
- 15. The method of claim 14, wherein the stack of layered membranes comprises 20 or more membranes.
- 16. The method of claim 14, wherein the stack of layered membranes comprises 50 or more membranes.
- 17. The method of claim 1, wherein the sample comprises a nucleic acid, a protein, a lipid, a carbohydrate, or a combination or mixture thereof.
- 18. The method of claim 1, wherein the sample is a substantially two-dimensional sample.
- 19. The method of claim 18, wherein the substantially two-dimensional sample is selected from the group consisting of a tissue section, a tissue microarray, a LCM harvested sample, a “one-dimensional” electrophoretic gel, a “two-dimensional” electrophoretic gel, a structurally transformed sample, or a combination of two or more thereof.
- 20. The method of claim 1, further comprising correlating the biomolecules detected on the one or more membranes with a biological characteristic of the sample.
- 21. A method of making multiple substantial copies of a biological sample, comprising:
providing a stack of layered membranes, wherein the membranes permit biomolecules applied to the stack to move through a plurality of the membranes, while directly capturing at least a portion of the biomolecules on multiple membranes; and applying the biological sample to the stack, under conditions that allow the multiple membranes to directly capture the biomolecules from the sample and form the multiple substantial copies of the biological sample, thereby making multiple substantial copies of the biological sample.
- 22. The method of claim 21, wherein the biological sample comprises a nucleic acid, a protein, a lipid, a carbohydrate, or a combination or mixture thereof.
- 23. The method of claim 21, wherein the biological sample is selected from the group consisting of a tissue section, a tissue microarray, a LCM harvested sample, a “one-dimensional” electrophoretic gel, a “two-dimensional” electrophoretic gel, a structurally transformed sample, or a combination of two or more thereof.
- 24. The method of claim 21, further comprising detecting one or more biomolecules of interest on at least one of the multiple substantial copies.
- 24. The method of claim 24, wherein detecting biomolecules of interest comprises exposing a plurality of the multiple membranes to at least one detector molecule.
- 25. The method of claim 24, wherein the biological sample is a tissue specimen that is placed on the stack of layered membranes, and biomolecules from the tissue specimen are directly captured by the layered membranes as the biomolecules from the tissue specimen move through the multiple membranes.
- 26. The method of claim 24, further comprising separating the multiple membranes prior to detecting the biomolecules of interest.
- 27. The method of claim 24, wherein the biomolecules applied to the stack themselves comprise detectors that are exposed to a biological specimen to be analyzed, and the method further comprises exposing one or more of the multiple membranes to the biological specimen under conditions that allow the biological specimen to be analyzed by the detectors.
- 28. The method of claim 27, wherein at least one biomolecule of interest on the multiple membranes is a nucleic acid molecule, and detecting biomolecules of interest comprises exposing the nucleic acid molecules on the multiple membranes to the biological specimen to be analyzed, under conditions that allow hybridization between the nucleic acid molecules on the membranes and nucleic acid molecules in the biological specimen.
- 29. A method of creating a set of microarray copies comprising:
providing a stack of layered membranes; and applying a plurality of DNA probes, antibodies, or a combination thereof, to the stack of layered membranes, wherein the stack of layered membranes comprises a plurality of substrates through which the probes or antibodies move, and in which a portion of the probes or antibodies are directly captured by one or more of the substrates.
- 30. The method of claim 29, further comprising separating the substrates to provide corresponding substrates having a plurality of the DNA probes, antibodies or combination thereof, in corresponding positions of each of the substrates.
- 31. The method of claim 29, wherein applying the plurality of DNA probes, antibodies, or combination thereof, is applied to the stack from a plate having a plurality of wells each containing a different DNA probe or antibody, and the DNA probes or antibodies are transferred from the wells to the stack so as to create a set of substantially replicate microarrays.
- 32. A method of analyzing biomolecules in a tissue sample, comprising:
providing at least one membrane; positioning the at least one membrane in contact with the tissue sample; applying heat and/or pressure to the tissue sample, whereupon biomolecules are transferred from the tissue sample onto the at least one membrane; and, analyzing the biomolecules on the at least one membrane.
- 33. The method of claim 32, wherein the tissue sample is an archival tissue sample, a cryo-preserved tissues ample, a fresh tissue sample, an LCM-harvested tissue sample, or a tissue microarray.
- 34. The method of claim 32, comprising providing a plurality of membranes and further comprising analyzing the biomolecules on two or more of the plurality of membranes.
- 35. The method of claim 32, wherein the membrane is a porous membrane of no more than 30 microns thickness, comprising a core substrate and a coating.
- 36. The method of claim 35, wherein the core substrate comprises polycarbonate.
- 37. The method of claim 35, wherein the coating comprises nitrocellulose.
- 38. A method of replicating biomolecular content of a tissue microarray, comprising:
providing the tissue microarray; and transferring biomolecules from the tissue microarray onto a plurality of membranes so as to produce at least one replicate of the biomolecular content of the tissue microarray.
- 39. The method of claim 38, wherein transferring biomolecules comprises:
positioning the plurality of membrane in contact with the tissue microarray; and applying heat and/or pressure to the tissue microarray, whereupon biomolecules are transferred from the tissue microarray onto at least one membrane of the plurality of membranes.
- 40. A method of analyzing cellular material embedded on an LCM transfer film comprising:
providing one or more membranes; positioning the one or more membranes adjacent to the LCM transfer film; transferring biomolecules from the cellular material to the one or more membranes; and detecting the biomolecules on the membranes.
- 41. The method of claim 40, wherein transferring biomolecules comprises:
applying heat and/or pressure to the membranes and/or the LCM transfer film, whereupon biomolecules are transferred from the LCM transfer onto the one or more membranes.
- 42. A method for analyzing the proteome of a biological sample comprising:
separating at least one protein from another protein present in the biological sample; transferring a portion of the separated protein to a plurality of membranes in a stacked configuration; incubating each of the membranes in the presence of one or more species of predetermined ligand molecules under conditions sufficient to permit binding between the separated protein and a ligand capable of binding to such protein; and analyzing the proteome by determining the occurrence of binding between the protein and any of the species of predetermined ligand molecules.
- 43. The method of claim 42, wherein separating the at least one protein from another protein present in the sample comprises electrophoresis.
- 44. The method of claim 43, wherein the electrophoresis is two-dimensional gel electrophoresis.
- 45. The method of claim 42, wherein the sample is obtained from mammalian cells or tissue.
- 46. The method of claim 45, wherein the mammal is a human.
- 47. The method of claim 42, wherein transferring of a portion of the separated protein comprises gel transfer.
- 48. The method of claim 42, wherein transferring a portion of the separated protein comprises contact transfer.
- 49. The method of claim 42, wherein the mammalian cells or tissue are human cells or tissue.
- 50. The method of claim 42, wherein the separated protein is a product of a human gene.
- 51. The method of claim 42, wherein at least one of the species of ligand is selected from the group consisting of an antibody, an antibody fragment, a single chain antibody, a receptor protein, a solubilized receptor derivative, a receptor ligands, a metal ion, a virus, a viral protein, an enzyme substrate, a toxin, a toxin candidate, a pharmacological agent, and a pharmacological agent candidate.
- 52. The method of claim 51, wherein at least one of the species of ligand is an antibody or an antibody fragment.
- 53. The method of claim 51, wherein at least one of the species of ligand is a receptor protein, a solubilized receptor derivative, or a receptor ligand.
- 54. The method of claim 51, wherein at least one of the species of ligand is a pharmacological agent or pharmacological agent candidate.
- 55. The method of claim 51, wherein the binding of at least one of the species of ligand is dependent upon the structure of the separated protein.
- 56. The method of claim 51, wherein the binding of at least one of the species of ligand is dependent upon the biological function of the separated protein.
- 57. The method of claim 42, wherein at least one of the membranes is incubated with more than one species of ligand.
- 58. The method of claim 42, wherein the plurality of membranes comprises at least two membranes.
- 59. The method of claim 58, wherein the plurality of membranes comprises at least 10 membranes.
- 60. The method of claim 58, wherein the plurality of membranes comprises at least 20 membranes.
- 61. The method of claim 42, wherein the one or more ligand species comprises at least two ligand species.
- 62. The method of claim 61, wherein the one or more ligand species comprises at least 10 ligand species.
- 63. The method of claim 61, wherein the one or more ligand species comprises at least 20 ligand species.
- 64. The method of claim 42, wherein incubating each of the membranes is performed before separating at least one protein.
- 65. A method for uniquely visualizing a desired predetermined protein if present in a biological sample, the method comprising:
separating proteins present in the sample from one another; transferring a portion of the separated proteins of the sample to a plurality of membranes in a stacked configuration; incubating each of the membranes in the presence of one or more species of predetermined ligand molecules under conditions sufficient to permit binding between desired predetermined protein and a ligand capable of binding to such protein; and visualizing any binding between the protein and any of the species of predetermined ligand molecules.
- 66. A method for identifying biomolecules that have been separated on a solid support, the method comprising:
contacting a stack of membranes to the solid support containing the separated biomolecules; permitting biomolecules to be transferred from the solid support to multiple membranes in the stack; separating the membranes from the stack; and identifying one or biomolecules transferred to at least one of the membranes.
- 67. The method of claim 66, wherein the method is a method for identifying proteins, and the biomolecules comprise proteins.
- 68. The method of claim 66, wherein the membranes have a high affinity but a low capacity for at least one class of biomolecule.
- 69. The method of claim 67, wherein the membranes have a high affinity but a low capacity for proteins.
- 70. The method of claim 66, wherein at least some of the biomolecules are transferred to each membrane of the stack.
- 71. The method of claim 66, wherein permitting the biomolecules to be transferred from the support to multiple membranes in a stack produces multiple replicate membranes.
- 72. The method of claim 66, wherein the biomolecules are separated on a gel.
- 73. The method of claim 72, wherein the separation comprises electrophoresis.
- 74. The method of claim 73, wherein the electrophoresis is SDS PAGE.
- 75. The method of claim 74, wherein more than 30 micrograms of protein is loaded into a well of the gel.
- 76. The method of claim 75, wherein about 50 to about 100 micrograms of protein are loaded into a well of the gel.
- 77. The method of claim 69, wherein the membranes comprise polycarbonate.
- 78. The method of claim 69, wherein the membranes comprise a cellulose derivative.
- 79. The method of claim 78, wherein the cellulose derivative is cellulose acetate.
- 80. The method of claim 66, wherein the membranes comprise a polyolefin.
- 81. The method of claim 66, wherein the stack comprises at least 5 membranes.
- 82. The method of claim 81, wherein the stack comprises at least 10 membranes.
- 83. The method of claim 66, wherein each membrane is less than about 30 microns thick.
- 84. The method of claim 83, wherein each membrane is about 8 to 10 microns thick.
- 85. The method of claim 69, wherein at least one side of the membranes is treated to increase specific binding of proteins.
- 86. The method of claim 85, wherein at least one side of the membranes is treated to increase specific binding of the proteins or other targeted proteins.
- 87. The method of claim 85, wherein the treatment comprises a coating of nitrocellulose.
- 88. The method of claim 85, wherein the treatment comprises a coating of poly-L-lysine.
- 89. The method of claim 66, wherein the membranes are in a frame, the frame being mounted to the periphery of the membranes, wherein the frame defines a channel for passing fluids or air away from the space intermediate the membrane and an adjacent membrane.
- 90. A method for identifying biomolecules that have been separated on a solid support, the method comprising:
providing a solid support containing the separated biomolecules wherein the support has an upper side and a lower side; applying a first stack of membranes to the upper side and a second stack of membranes to the lower side; permitting the biomolecules to be transferred from the support to the first and second membrane stacks; separating the membranes, and identifying one or more biomolecules transferred to at least one of the membranes.
- 91. The method of claim 90, wherein the biomolecules comprise proteins.
- 92. A kit comprising:
a membrane array for detecting biomolecules in a sample, the array comprising a plurality of membranes, wherein each of the plurality of membranes has substantially a same affinity for the biomolecules; and containers of detector molecules for detecting biomolecules captured on each membrane.
- 93. The kit of claim 92, wherein the detector molecules are antibodies or probes.
- 94. The kit according to claim 92 wherein the membranes comprise a polymer substrate coated with a material for increasing an affinity of the substrate to the biomolecules.
- 95. The kit according to claim 94 wherein the coating material comprises nitrocellulose.
- 96. The kit according to claim 93, wherein the antibodies or probes are specific capture molecules for biomolecules sought to be detected on particular membranes of the array.
- 97. The kit according to claim 96 wherein each container contains an antibody cocktail, and each antibody cocktail comprises at least two antibodies with different binding specificity.
- 98. The kit according to claim 92 wherein the plurality of membranes has a low capacity for the biomolecules.
- 99. The kit according to claim 92 wherein the plurality of membranes each have a thickness of less than about 30 microns.
- 100. The kit according to claim 99, wherein the plurality of membranes comprise a core substrate comprising polycarbonate, and a coating comprising nitrocellulose.
- 101. A kit for comparing the molecular profiles of tissue samples, comprising:
at least one tissue microarray; and at least one replicate of the tissue microarray.
- 102. The kit of claim 101, wherein the at least one replicate of the tissue microarray was produced using the method of claim 38.
- 103. A kit for replicating a pattern of biomolecules from a tissue sample, comprising:
a plurality of membranes, each having a coating on its upper and/or lower surfaces to increase specific binding of a target biomolecule; a quantity of transfer buffer; and a fluid impervious enclosure.
- 104. The kit of claim 103, further comprising instructions for carrying out the method of claim 32.
- 105. A kit for analyzing a proteome comprising:
a plurality of membranes, each having a affinity for at least one protein; and a plurality of reagent species, each adapted to detect one or more specific proteins bound to the membranes.
- 106. The kit of claim 105, further comprising instructions setting forth the particular groups of reagents to be applied to each of the membranes.
- 107. The kit of claim 104, wherein the membranes comprise a porous substrate having a thickness of less than about 30 microns.
- 108. The kit of claim 107, wherein the membranes are polycabonate membranes, coated with a material for increasing the affinity of the membrane to biomolecules.
- 109. The kit of claim 108, wherein the membranes are coated with nitrocellulose.
- 110. The kit according to claim 105 wherein the reagent species are selected from the group consisting of an antibody, an antibody fragment, a single chain antibody, a receptor protein, a solubilized receptor derivative, a receptor ligands, a metal ion, a virus, a viral protein, an enzyme substrate, a toxin, a toxin candidate, a pharmacological agent, and a pharmacological agent candidate.
- 111. A kit for uniquely visualizing a desired predetermined protein if present in a biological sample, comprising:
a plurality of membranes, each having a specific affinity for at least one protein, and a plurality of reagent species, each adapted to detect the desired predetermined protein if bound to the membranes.
- 112. A membrane unit for blotting comprising:
a stack of at least two porous membranes having a thickness no greater than about 30 microns; and a frame, mounted to the membranes, the frame a having a thickness no greater than about 300 microns.
- 113. The membrane unit of claim 112, wherein the unit comprises more than two membranes.
- 114. The membrane unit of claim 112, wherein the unit comprises a frame, mounted to the periphery of the membranes, wherein the frame defines a channel for passing fluids or air away from the space intermediate the membrane and an adjacent membrane.
- 115. The membrane unit of claim 112, wherein the membranes have a high affinity but a low capacity for proteins.
- 116. The membrane unit of claim 115, wherein the membranes comprise polycarbonate.
- 117. The membrane unit of claim 115, wherein the membranes comprise a cellulose derivative.
- 118. The membrane unit of claim 117, wherein the cellulose derivative is cellulose acetate.
- 119. The membrane unit of claim 115, wherein the membranes comprise a polyolefin.
- 120. The membrane unit of claim 112, wherein the stack comprises at least 5 membranes.
- 121. The membrane unit of claim 120, wherein the stack comprises at least 10 membranes.
- 122. The membrane unit of claim 112, wherein the thickness of the membranes is less than about 30 microns.
- 123. The membrane unit of claim 122, wherein the thickness of the membrane is about 8 to 10 microns.
- 124. The membrane unit of claim 112, wherein at least one side of the membranes is treated to increase specific binding of a biomolecule.
- 125. The membrane unit of claim 115, wherein at least one side of the membranes is treated to increase specific binding of the proteins or other targeted proteins.
- 126. The membrane unit of claim 125, wherein the treatment comprises a coating of nitrocellulose.
- 127. The membrane unit of claim 115, wherein the treatment comprises a coating of poly-L-lysine.
- 128. A membrane for use in the membrane unit of claim 112.
- 129. The membrane of claim 128, wherein at least one side of the membranes is treated to increase specific binding of a targeted biomolecule.
- 130. A porous membrane having a high affinity but low capacity for biomolecules, the membrane comprising a core substrate and a coating, wherein the membrane has a thickness of no more than about 30 microns.
- 131. The membrane of claim 130, wherein the core substrate comprises polycarbonate, cellulose acetate, a polyolefin, or combinations of two or more thereof.
- 132. The membrane of claim 130, wherein the coating comprises nitrocellulose, poly-L-lysine, or mixtures thereof.
- 133. The membrane of claim 130, wherein the core substrate comprises polycarbonate and the coating comprises nitrocellulose.
- 134. The membrane of claim 130, wherein the membrane has a thickness of about 8-10 microns.
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation in Part (CIP) of U.S. patent application Ser. No. 09/753,574 (filed Jan. 4, 2001), which is a CIP of U.S. patent application Ser. No. 09/718,990 (Filed Nov. 20, 2000), which is a CIP of International Patent Application No. US00/20354, filed Jul. 26, 2000 and published in the English language, and claims the benefit of U.S. Provisional Patent Application No. 60/145,613 (filed Jul. 26, 1999). The current application further claims the benefit of U.S. Provisional Patent Application Nos. 60/286,258 (filed Apr. 25, 2001), 60/304,031 (filed Jul. 9, 2001), and 60/296,475 (filed Jun. 8, 2001). Each of these related applications is incorporated herein in their entirety.
STATEMENT OF GOVERNMENT RIGHTS
[0002] At least one of the inventors is an employee of an agency of the Government of the United States, and the Government may have certain rights in this invention.
PCT Information
Filing Document |
Filing Date |
Country |
Kind |
PCT/US01/44009 |
11/20/2001 |
WO |
|