Difference detection methods using matched multiple dyes
First Claim
1. A method of comparing protein compositions between at least two different samples comprising:
- (a) preparing an extract of proteins from each of said at least two samples;
(b) providing a set of matched luminescent dyes chosen from dyes capable of covalently binding to proteins within said extract of proteins, wherein each dye within said set (1) has a net charge which will maintain the overall net charge of the proteins upon such covalent binding and has ionic and pH characteristics whereby relative migration of a protein labeled with any one of said dyes is the same as relative migration of said protein labeled with another dye in said set, (2) emits luminescent light at a wavelength that is sufficiently different from the emitted luminescent light of remaining dyes in said set to provide a detectably different light signal;
(c) reacting each extract of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins;
(d) mixing each of said dye labeled proteins to form a single mixture of different dye-labeled proteins;
(e) separating the dye-labeled proteins of interest within said mixture; and
(f) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by;
capturing images of the dye-labeled proteins at different wavelengths of emitted luminescence; and
processing the images to determine the difference in luminescent intensity.
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Abstract
A process and a kit are provided for detecting differences in two or more samples of protein, including proteins bearing post-translational modifications and peptides. Proteins are prepared, for example, from each of a different group of cell samples or body fluid samples to be compared. Each protein extract is labeled with a different one of a luminescent dye from a matched set of dyes. The matched dyes have generally the same ionic and pH characteristics but emit light at different wavelengths to exhibit a different color upon luminescence detection. The labeled protein extracts are mixed together and separated together by electrophoresis or a chromatographic method. The separation is observed to detect proteins unique to one sample or present in a greater ratio in one sample than in the other. Those unique or excess proteins will fluoresce the color of one of the dyes used. Proteins common to each sample migrate together and fluoresce the same.
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Citations
37 Claims
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1. A method of comparing protein compositions between at least two different samples comprising:
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(a) preparing an extract of proteins from each of said at least two samples;
(b) providing a set of matched luminescent dyes chosen from dyes capable of covalently binding to proteins within said extract of proteins, wherein each dye within said set (1) has a net charge which will maintain the overall net charge of the proteins upon such covalent binding and has ionic and pH characteristics whereby relative migration of a protein labeled with any one of said dyes is the same as relative migration of said protein labeled with another dye in said set, (2) emits luminescent light at a wavelength that is sufficiently different from the emitted luminescent light of remaining dyes in said set to provide a detectably different light signal;
(c) reacting each extract of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins;
(d) mixing each of said dye labeled proteins to form a single mixture of different dye-labeled proteins;
(e) separating the dye-labeled proteins of interest within said mixture; and
(f) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by;
capturing images of the dye-labeled proteins at different wavelengths of emitted luminescence; and
processing the images to determine the difference in luminescent intensity. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method of comparing protein compositions between at least two different samples comprising:
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(a) preparing an extract of proteins from each of said at least two samples;
(b) providing a set of matched luminescent dyes chosen from dyes capable of covalently binding to proteins within said extract of proteins, wherein each dye within said set (1) has a net charge which will maintain the overall net charge of the proteins upon such covalent binding and has ionic and pH characteristics whereby relative migration of a protein labeled with any one of said dyes is the same as relative migration of said protein labeled with another dye in said set, (2) emits luminescent light at a wavelength that is sufficiently different from the emitted luminescent light of remaining dyes in said set to provide a detectably different light signal;
(c) reacting each extract of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins;
(d) mixing each of said dye labeled proteins to form a single mixture of different dye-labeled proteins;
(e) separating the dye-labeled proteins of interest within said mixture; and
(f) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by;
capturing a first image of the dye labeled proteins using a first filter or filters that only allows the passage of light having the wavelength of the luminescent light emitted by a first dye used in labeling the proteins of interest;
capturing a second image of the dye labeled proteins using a second filter or filters that only allows the passage of light having the wavelength of the luminescent light emitted by a second dye used in labeling the proteins of interest; and
processing the first and second images to determine the difference in luminescent intensity. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A method of comparing protein compositions between at least two different samples comprising:
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(a) preparing an extract of proteins from each of said at least two samples;
(b) providing a set of matched luminescent dyes chosen from dyes capable of covalently binding to proteins within said extract of proteins, wherein each dye within said set (1) has a net charge which will maintain the overall net charge of the proteins upon such covalent binding and has ionic and pH characteristics whereby relative migration of a protein labeled with any one of said dyes is the same as relative migration of said protein labeled with another dye in said set, (2) emits luminescent light at a wavelength that is sufficiently different from the emitted luminescent light of remaining dyes in said set to provide a detectably different light signal;
(c) reacting each extract of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins;
(d) mixing each of said dye labeled proteins to form a single mixture of different dye-labeled proteins;
(e) placing said mixture in an electrophoresis gel and separating the dye-labeled proteins of interest within said mixture;
(f) making images of the gel; and
,(g) processing the images with a computer to detect the difference in luminescent intensity between the different dye-labeled proteins of interest. - View Dependent Claims (24)
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25. A method of comparing protein compositions between at least two different samples comprising:
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(a) preparing an extract of proteins from each of said at least two samples;
(b) providing a set of matched luminescent dyes chosen from dyes capable of covalently binding to proteins within said extract of proteins, wherein each dye within said set (1) has a net charge which will maintain the overall net charge of the proteins upon such covalent binding and has ionic and pH characteristics whereby relative migration of a protein labeled with any one of said dyes is the same as relative migration of said protein labeled with another dye in said set, (2) emits luminescent light at a wavelength that is sufficiently different from the emitted luminescent light of remaining dyes in said set to provide a detectably different light signal;
(c) reacting each extract of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins;
(d) mixing each of said dye labeled proteins to form a single mixture of different dye-labeled proteins;
(e) separating the dye-labeled proteins of interest within said mixture;
(f) capturing images of said separated dye-labeled proteins; and
,(g) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by computer analysis of the images. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32)
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33. A method of comparing protein compositions between at least two different samples comprising:
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(a) preparing an extract of proteins from each of said at least two samples;
(b) providing a set of matched luminescent dyes chosen from dyes capable of covalently binding to proteins within said extract of proteins, wherein each dye within said set (1) has a net charge which will maintain the overall net charge of the proteins upon such covalent binding and has ionic and pH characteristics whereby relative migration of a protein labeled with any one of said dyes is the same as relative migration of said protein labeled with another dye in said set, (2) emits luminescent light at a wavelength that is sufficiently different from the emitted luminescent light of remaining dyes in said set to provide a detectably different light signal;
(c) reacting each extract of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins;
(d) mixing each of said dye labeled proteins to form a single mixture of different dye-labeled proteins;
(e) separating the dye-labeled proteins of interest within said mixture; and
(f) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by;
capturing luminescent data for the dye-labeled proteins at different wavelengths of emitted luminescence; and
processing said data to determine the difference in luminescent intensity. - View Dependent Claims (34, 35, 36, 37)
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Specification