Source: https://patents.google.com/patent/US20060211011
Timestamp: 2018-02-19 02:33:35
Document Index: 676432654

Matched Legal Cases: ['§371', 'application No. 60', 'application No. 60', '§371', 'application No. 60', 'art 2002']

US20060211011A1 - Methods, devices and kits for multiplex blotting of biological samples from multi-well plates - Google Patents
Methods, devices and kits for multiplex blotting of biological samples from multi-well plates
US20060211011A1
US20060211011A1 US11277227 US27722706A US2006211011A1 US 20060211011 A1 US20060211011 A1 US 20060211011A1 US 11277227 US11277227 US 11277227 US 27722706 A US27722706 A US 27722706A US 2006211011 A1 US2006211011 A1 US 2006211011A1
US11277227
US7838222B2 (en )
Dan-Paul Hartmann
GOVERNMENT OF UNITED STATES OF AMERICA/NIH
Emmert-Buck Michael R
This is a continuation-in-part of co-pending U.S. application Ser. No. 10/522,663, which is a §371 U.S. national stage of PCT/US2003/024225, filed Aug. 1, 2003, which claims the benefit of U.S. provisional application No. 60/400,874, filed Aug. 2, 2002; and a continuation-in-part of co-pending U.S. application Ser. No. 10/627,352, filed Jul. 25, 2003, which is a continuation of U.S. application Ser. No. 09/718,990, filed Nov. 20, 2000 and now issued as U.S. Pat. No. 6,602,661, which in turn is a continuation-in-part of PCT/US00/20354, filed Jul. 26, 2000, which claims the benefit of U.S. provisional application No. 60/145,613 filed Jul. 26, 1999; and further is a continuation-in-part of co-pending U.S. application Ser. No. 10/048,194, which is a §371 U.S. national stage of PCT/US00/20354, filed Jul. 26, 2000, which also claims the benefit of U.S. provisional application No. 60/145,613, filed Jul. 26, 1999. Each of the applications from which priority and/or benefit is claimed is incorporated herein in its entirety.
FIG. 4 is an image of a Western blot of a cellular extract made from human epithelial cell line A431 treated with P13 kinase inhibitor LY294002 (row marked “INHIBITOR”) and control A431 cells which were untreated (row marked “CONTROL”). Clarified total cell extracts were transferred to a stack of ten membranes according to a method described herein. The membrane shown was then probed with an antibody specific for the phosphorylated form of the Akt protein.
“Fluorophore” refers to a chemical compound, which when excited by exposure to a particular wavelength of light, emits light (i.e., fluoresces), for example at a different wavelength. Fluorophores can be described in terms of their emission profile, or “color.” Green fluorophores, for example Cy3, FITC, and Oregon Green, are characterized by their emission at wavelengths generally in the range of 515-540 λ. Red fluorophores, for example Texas Red, Cy5 and tetramethylrhodamine, are characterized by their emission at wavelengths generally in the range of 590-690 λ.
The substrates are preferably “track-etched membranes” (a/k/a “screen membranes”), which are formed by a process that creates well-defined pores by exposing a dense film to ionizing radiation forming damage tracks. This is followed by etching of the damaged tracks into pores by a strong alkaline solution. A description of this process may be found on the Internet site of G.E. Osmonics (Minnetonka, Minn.) under the heading “Basic Principles of Microfiltration.” Examples of membranes that may be employed as the substrate include the Isopore™ (polycarbonate film) membrane available from Millipore (Bedford, Mass.), the Poretics® Polycarbonate or Polyester membranes available from Osmonics (Minnetonka, Minn.) or the Cyclopore™ Polycarbonate or Polyester membranes available from Whatman (Clifton, N.J.).
In one embodiment, the materials that comprise frames 50 should be able to maintain their structure at temperatures of up to 80° C., but be able to melt when applied to a typical heat-sealing apparatus. One skilled in the relevant art will readily appreciate that a variety of compositions and configurations of frames 50 could meet these requirements. Examples of materials that may be employed to make frames 50 are transparency film (such as that available from Canon), or any thin plastic sheet made of polycarbonate, polyester or polyvinylchloride.
Another approach to identifying protein function is to first renature the proteins on the membranes by any of a number of techniques known in the art, for instance incubating the membrane in Triton-X® (octylphenol polymerized with ethylene oxide). Once renatured, proteins will regain their enzymatic activity and one of several substrate degradation assays known in the art can be used. With this approach the activity of classes of proteins, for instance, kinases, phosphates and metalloproteinases, can be determined on the membranes using known techniques.
Software may also be provided for use with methods, devices or kits described herein, for instance to analyze and/or overlay the images of spots (features) produced on the membranes after transfer. The software is adapted to quantify the biomolecules (e.g., proteins or nucleic acids) by determining the density of the spot/feature (for instance, based on the intensity of signal from a label) and comparing these densities to a control. This process is known as “normalization.” A variety of commercially available programs may be employed, such as, the ID Image Analysis Software available from Eastman Kodak Co. or Image Quant Software available from Amersham Biosciences. The software preferably allows the user to select the kind of comparative analysis to be performed ( for instance, comparing the spots present in one sample with those in another sample, or comparing the spots present on one membrane with those of another membrane within the same membrane stack). Results of the analysis is displayed in tabular format and user is given the option to go back and compare magnified sections of the images of interest.
EXAMPLE 1 Transfer from Multi-Well Plate to Membranes
A Hybri-Slot 24-Well Filtration Manifold apparatus from Invitrogen (Carlsbad, Calif.) was used in this example. Ten membranes were sized to 12 cm×3 cm and soaked in ddH2O for 2 minutes. The ten membranes were then placed between the top and bottom plate of the vacuum manifold. The apparatus was assembled in accordance with the manufacturer's recommendations.
BSA 0 0.002 0.01 0.02 0.04 0.08 0.12 0.16 0.2 0.24 0.28 0.32
Sample 50 50 50 50 50 50 50 50 50 50 50 50
BSA 0 0.1 0.5 1 2 4 6 8 10 12 14 16
EXAMPLE 2 Detection of Specific Proteins
Human epithelial cell line A431 (vulvar squamous carcinoma origin) was maintained at 37° C. and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (Life Technologies, Inc.). To analyze signal transduction in the absence of growth factors, cells were grown to 50-60% confluency in 6-well culture plates (#3046, Falcon, Lincoln Park N.J.). Once a desired cell density was reached, the cells were washed twice in sterile Dulbecco's phosphate-buffered saline (DPBS) and incubated for 24 hours in DMEM containing 0.1% FBS, in order to induce quiescence.
Quiescent cells were incubated for 2 hours with various concentrations of the P13 kinase inhibitor LY294002 (2-(4-morpholinyl)-8-phenyl-chromone; Cell Signaling) ranging from between 2 to 50 μM. Complete inhibition of Akt kinase activity was achieved at concentrations of LY294002 between 10 to 50 μM, while only partial inhibition was achieved with lesser concentrations (e.g., 2 and 5 μM).
Following incubation with the inhibitor, the treated cells were washed twice in cold DPBS and extraction buffer (3M KCl, 10 mM TRIS pH 7.5, 100 mM NaCl, 50 mM EDTA; 1:100 dilution of phosphatase-inhibitor cocktails I and II (Sigma)) was added to achieve a density of 1×105 cells/ml. The cells were scraped off the tissue culture plates, transferred into microfuge tubes and vortexed for 3 minutes. After 30 minutes incubation at room temperature with occasional vortexing, the tubes were centrifuged at 14,000 RPM for 4 minutes and the supernatants were separated into fresh tubes.
One of the membranes was incubated in 1:200 dilution of anti-phospho-Akt (S473) (Pharmigen) in TBST with 0.5% BSA at 4° C. for 8-12 hours. The membrane was washed 3 times for 5 minutes each in TBST and incubated in a 1:2,000 dilution of anti-rabbit secondary antibody (Amersham) for 30 minutes at room temperature. Following incubation with the secondary antibody, the membrane was washed 3 times for 5 minutes each in TBST. Phospho-Akt transferred to the membrane was visualized using ECL Plus Reagent (Amersham) and BioMax film (Kodak).
EXAMPLE 3 Simultaneous Detection of Total and Specific Proteins
14. The method of claim 20, wherein the conditions that allow at least a portion of the samples to pass through the multiple membranes comprise applying at least partial vacuum that encourages movement of the sample through the stack of membranes in a desired direction of movement.
15. The method of claim 20, wherein the conditions that allow at least a portion of the samples to pass through the multiple membranes comprise providing a wick that facilitates movement of the sample through the stack of membranes in a desired direction of movement.
16. The method of claim 20, wherein the stack of membranes comprises 5 or more membranes.
17. The method of claim 20, wherein at least one of the samples comprises nucleic acid.
18. The method of claim 17, wherein the at least one sample comprises DNA.
19. The method of claim 20, further comprising correlating the biomolecules detected on the one or more membranes with a biological characteristic of the sample.
20. A method for identifying one or more biomolecules from a plurality of wells in a multi-well plate comprising:
providing a stack of at least two membranes;
introducing biomolecules into two or more wells of the multi-well plate;
transferring biomolecules from the wells to multiple membranes in the stack;
separating the membranes; and
identifying the one or more biomolecules on the multiple membranes.
21. The method of claim 20, wherein the biomolecules comprise proteins, DNA molecules, RNA molecules, or mixtures of two or more thereof.
22. The method of claim 20, wherein the membranes are formed of track-etched polymeric material.
23. The method of claim 22, wherein the membranes are formed of track-etched polycarbonate.
24. The method of claim 23, wherein one or more of the membranes are coated with an antibody or other capture molecule having an affinity to a particular target molecule.
25. The method of claim 20, wherein one or more of the membranes are coated with an antibody or other capture molecule having an affinity to a particular target molecule.
26. The method of claim 25, wherein the membranes are formed of track-etched polycarbonate.
27. A method of making multiple substantial replicas of a biomolecular content of a multi-well sample holder, which method comprises:
providing a stack of membranes, wherein the membranes are formed of track-etched polymeric material and one or more membranes are coated with an antibody or other capture molecule having an affinity to a particular target molecule;
applying two or more samples comprising biomolecules to at least two wells of the multi-well sample holder, under conditions that
(a) allow at least a portion of the samples to pass through the stack of membranes; and
(b) allow the multiple membranes to capture at least a portion of the biomolecules from each of the samples, thereby forming multiple substantial replicas of the biomolecular content of the multi-well sample holder.
28. A method of analyzing biomolecules in a biological sample, comprising the steps:
providing a plurality of stacked track-etched polymeric membranes which are separable;
transferring said biomolecules through said membranes and capturing a portion of said biomolecules on one or more of said membranes;
separating said membranes; and
analyzing one or more of the captured biomolecules.
29. The method of claim 28, further including the steps of providing a multi-well sample holder and receiving multiple biological samples therein for analyzing.
30. The method of claim 28, wherein one or more of the membranes are coated with an antibody or other capture molecule having an affinity to a target molecule in the biological sample.
31. The method of claim 28, wherein the membranes are polycarbonate.
32. The method of claim 29, wherein the membranes are polycarbonate.
33. The method of claim 30, wherein the membranes are polycarbonate.
34. The method of claim 33, wherein at least one of the membranes is coated with a peptide.
35. The method of claim 24, wherein at least one of the membranes is coated with a peptide.
US11277227 1999-07-26 2006-03-22 Methods, devices and kits for multiplex blotting of biological samples from multi-well plates Active 2022-12-08 US7838222B2 (en)
US14561399 true 1999-07-26 1999-07-26
PCT/US2000/020354 WO2001007915A8 (en) 1999-07-26 2000-07-26 Layered device with capture regions for cellular analysis
US09718990 US6602661B1 (en) 1999-07-26 2000-11-20 Methods and arrays for detecting biomolecules
US4819402 true 2002-02-15 2002-02-15
US40087402 true 2002-08-02 2002-08-02
US10627352 US20040081987A1 (en) 1999-07-26 2003-07-25 Methods and arrays for detecting biomolecules
US10522663 US20050255473A1 (en) 2002-08-02 2003-08-01 Methods, devices and kits for multiplex blotting of biological samples from multi-well plates
PCT/US2003/024225 WO2004013607A3 (en) 2002-08-02 2003-08-01 Methods, devices and kits for multiplex blotting of biological samples from multi-well plates
US11277227 US7838222B2 (en) 1999-07-26 2006-03-22 Methods, devices and kits for multiplex blotting of biological samples from multi-well plates
US12626405 US8779312B2 (en) 1999-07-26 2009-11-25 Method and device for analyzing biomolecules with track-etched polymeric layers
US10522663 Continuation-In-Part
US10048194 Continuation-In-Part
PCT/US2000/020354 Continuation-In-Part WO2001007915A8 (en) 1999-07-26 2000-07-26 Layered device with capture regions for cellular analysis
US4819402 Continuation-In-Part 2002-02-15 2002-02-15
US10627352 Continuation-In-Part US20040081987A1 (en) 1999-07-26 2003-07-25 Methods and arrays for detecting biomolecules
US10522663 Continuation-In-Part US20050255473A1 (en) 2002-08-02 2003-08-01 Methods, devices and kits for multiplex blotting of biological samples from multi-well plates
PCT/US2003/024225 Continuation-In-Part WO2004013607A3 (en) 2002-08-02 2003-08-01 Methods, devices and kits for multiplex blotting of biological samples from multi-well plates
US12626405 Division US8779312B2 (en) 1999-07-26 2009-11-25 Method and device for analyzing biomolecules with track-etched polymeric layers
US20060211011A1 true true US20060211011A1 (en) 2006-09-21
US7838222B2 US7838222B2 (en) 2010-11-23
ID=46324124
US11277227 Active 2022-12-08 US7838222B2 (en) 1999-07-26 2006-03-22 Methods, devices and kits for multiplex blotting of biological samples from multi-well plates
US12626405 Active 2021-04-02 US8779312B2 (en) 1999-07-26 2009-11-25 Method and device for analyzing biomolecules with track-etched polymeric layers
US (2) US7838222B2 (en)
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US8779312B2 (en) 2014-07-15 grant
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