Difference gel electrophoresis using matched multiple dyes

• US 6,043,025 A
• Filed: 10/10/1997
• Issued: 03/28/2000
• 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): SAUNDERS, DAVID A
• Application Number:
  US08/949,115
• Time in Patent Office: 900 Days
• Field of Search: 204/459, 204/461, 436/63, 436/86, 436/172, 436/800, 435/4, 435/810
• US Class Current: 435/4
• 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 kit for use in detecting the presence of differences in the protein content of at least two different samples comprising:

a matched set of luminescent dyes, each dye within said set being capable of covalently binding to protein, 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.

2. The kit recited in claim 1 further comprising at least one electrophoresis gel.

3. The kit recited in claim 1 further comprising materials to quench a labeling reaction between the dyes and protein in samples tested.

4. The kit recited in claim 1 wherein the 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, and CH₃ --C--CH₃, m is an integer from 1 to 3, one of R₁ and R₂ is a reactive group reactive with protein and the other is an alkyl, and wherein m differs for different dyes within said set of dyes and, optionally, the length of the alkyl differs for different dyes within said set of dyes to generally match the overall molecular weights of the different dyes within said set of dyes.

5. The kit recited in claim 1 wherein the luminescent dyes are derivatives of dipyrromethene boron difluoride dyes having positively charged linker groups to bind the dye to the protein.