Difference gel electrophoresis using matched multiple dyes

• US 6,127,134 A
• Filed: 04/20/1995
• Issued: 10/03/2000
• Est. Priority Date: 04/20/1995
• Status: Expired due to Term

• Current Assignee: Carnegie Mellon University
• Original Assignee: Carnegie Mellon University
• Inventors: Waggoner, Alan, Minden, Jonathan
• Primary Examiner(s): MINNIFIELD, NITA M
• Application Number:
  US08/425,480
• Time in Patent Office: 1,993 Days
• Field of Search: 435/47.2, 435/6, 436/800, 436/63, 436/86, 530/344, 530/412
• US Class Current: 435/7.2
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

A process and a kit are provided for detecting differences in two or more samples of protein. Protein extracts are prepared, for example, from each of a different group of cell 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 electrophoresed together. The gel 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.

Claims

1. A method of comparing protein composition of interest between at least two different cell samples comprising:

(a) preparing an extract of proteins from each of said at least two cell 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 electrophoretic migration of a protein labeled with any one of said dyes is the same as relative electrophoretic 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) quenching the labeling reactions of step (c) between the dyes and the protein;

(e) mixing each of said dye labeled proteins to form a single mixture of different dye-labeled protein;

(f) electrophoresing the mixture of step (e) by a predetermined electrophoresing method capable of separation within said mixture; and

(g) detecting the difference in luminescent intensity between the different dye-labeled protein by luminescent detection.

2. The method of claim 1 wherein said dyes bind to a primary amine of a lysine residue of the protein and each said dye within said luminescent dyes carries a net +1 charge.

3. The method of claim 1 wherein said set of matched luminescent dyes are cyanine dyes having the following structure:

##STR5## wherein the dotted lines each represent carbon atoms necessary for the formation of one to three fused rings having five to six atoms in each ring, X and Y are selected from the group consisting of S, O or CH₃ --C--CH₃, m is an integer from 1 to 3, one of R₁ and R₂ is a reactive group and the other is an alkyl.

4. The method of claim 3 wherein said reactive group reacts with an amino in the protein and is selected from the group consisting of isothiocyanate, (CH₂)₄ COOH and (CH₂)₄ CON-hydroxysuccinimidyl ester.

5. The method of claim 1 wherein said set of matched luminescent dyes are neutral dyes bound to the primary amino of a lysine residue in the protein by a positively charged linker group.

6. The method of claim 1 wherein said set of matched luminescent dyes are derivatives of dipyrromethene boron difluoride dyes having positively charged linker groups to bind the dye to the primary amino of a lysine residue in the protein.

7. The method of claim 1 wherein said dyes covalently bind to lysines in the proteins.

8. The method of claim 1 wherein said dyes covalently bind to sulfhydryl groups on the proteins.

9. The method of claim 1 wherein said dyes covalently bind to carboxylic acid groups on the proteins.

10. The method of claim 1 wherein the step of detecting differences in luminescent intensity is by fluorescent microscopy.

11. The method of claim 1 wherein the step of detecting differences in luminescent intensity is by electronic imaging.

12. The method of claim 1 wherein the proteins have binding sites for covalent binding to said dyes selected from the group consisting of lysine, carboxylic acid and sulfhydryl groups.

13. The method of claim 1 wherein the proteins binding sites for covalent binding to said dyes, and said binding sites are one of a moiety selected from the group consisting of lysine, carboxylic acid and sulfhydryl groups.

14. A method of comparing proteins of interest between at least two different samples comprising:

(a) preparing a mixture 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 said proteins within said mixture of proteins, wherein each dye within said set

(1) has ionic and pH characteristics whereby relative electrophoretic migration of a protein labeled with any one of said dyes is the same as relative electrophoretic 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 said mixtures of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled protein;

(d) quenching the labeling reactions of step (c) between the dyes and the proteins;

(e) mixing each of said dye labeled proteins to form a combined single mixture of different dye-labeled proteins;

(f) electrophoresing the combined mixture of step (e) by a predetermined electrophoresing method capable of separating the dye-labeled proteins of interest within said mixture; and

(g) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by luminescent detection.

15. The method of claim 14 wherein said set of matched luminescent dyes are cyanine dyes having the following structure:

##STR6## wherein the dotted lines each represent carbon atoms necessary for the formation of one to three fused rings having five to six atoms in each ring, X and Y are each selected from the group consisting of O, S and CH₃ --C--CH₃, m is an integer from 1 to 3, one of R₁ and R₂ is a reactive group and the other is an alkyl.

16. The method of claim 15 wherein said reactive group is selected from the group consisting of and isothiocyanate, (CH₂)₄ COOH and (CH₂)₄ CON-hydroxysuccinimidyl ester.

17. The method of claim 14 wherein said set of matched luminescent dyes are derivatives of dipyrromethene boron difluoride dyes having positively charged linker groups to bind the dye to the extract.

18. A method of comparing proteins of interest between at least two different samples comprising:

(a) preparing a mixture of proteins from each of said at least two samples;

(b) providing a set of matched luminescent dyes chosen from a single class of dyes capable of covalently binding to proteins within said mixture of proteins, wherein each dye within said set

(1) has ionic and pH characteristics whereby relative electrophoretic migration of a protein labeled with any one of said dyes is the same as relative electrophoretic 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 mixture of proteins of step (a) with a different dye from said set of step (b) to provide dye-labeled proteins;

(d) quenching the labeling reactions of step (c) between the dyes and the proteins;

(e) mixing each of said dye labeled proteins to form a combined single mixture of different dye-labeled proteins;

(f) electrophoresing the combined mixture of step (e) by a predetermined electrophoresing method capable of separating the dye-labeled proteins of interest within said combined mixture; and

(g) detecting the difference in luminescent intensity between the different dye-labeled proteins of interest by luminescent detection.