LABELING OF IMMOBILIZED PROTEINS USING DIPYRROMETHENEBORON DIFLUORIDE DYES

• US 20080182284A1
• Filed: 04/01/2008
• Published: 07/31/2008
• Est. Priority Date: 12/03/2001
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Martin, Karen J., Patton, Wayne F., Haugland, Richard P.
• Application Number:
  US12/060,834
• Publication Number:
  US 20080182284A1
• Time in Patent Office: Days
• US Class Current: 435/21
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/583 with non-fluorescent dye label
• CPC Class Codes: G01N 33/6839 involving dyes, e.g. Coomassie blue, bromcresol green

Abstract

The invention describes methods for labeling or detecting of immobilized poly(amino acids), including peptides, polypeptides and proteins, on membranes and other solid supports, using fluorescent dipyrrometheneboron difluoride dyes. Such immobilized poly(amino acids) are labeled or detected on blots or on arrays of poly(amino acids), or are attached to immobilized aptamers.

Claims

1. A kit for detection of poly(amino acids) immobilized on a solid surface, said kit comprising:

a. a dipyrrometheneboron difluoride dye of the formula;

2. The kit according to claim 1, further comprising a fluorogenic substrate.

3. The kit according to claim 2, wherein the specific binding pair member contains a label that is an enzyme and the enzyme is capable of utilizing the fluorogenic substrate to generate a detectable optical response.

4. The kit according to claim 1, wherein the specific binding pair member is an antibody or antibody fragment.

5. The kit according to claim 1, wherein the specific binding pair member contains a label that is a fluorescent dye.

6. The kit according to claim 1, wherein said specific binding pair member is a biotin-binding protein.

7. The kit according to claim 6, wherein the biotin-binding protein is avidin, Neutravidin or streptavidin.

8. The kit according to claim 1, wherein the label is an enzyme that is a peroxidase or a phosphatase.

9. The kit according to claim 8, wherein the peroxidase is horseradish peroxidase.

10. The kit according to claim 2, wherein the fluorogenic substrate is a peroxidase substrate that is a fluorescent tyramide.

11. The kit according to claim 8, wherein the phosphatase is alkaline phosphatase.

12. The kit according to claim 2, wherein the fluorogenic substrate is a phosphatase substrate that is a 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate.

13. The kit according to claim 2, wherein the fluorogenic substrate is a phosphatase substrate that is a 2-(5′

-chloro-2′

-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone.

14. The kit according to claim 2, wherein the fluorogenic substrate is a phosphatase substrate that is ELF 39 reagent.

15. The kit according to claim 1, wherein R¹ is methyl or -L-Rx;

R² is H, bromine, or -L-Rx;

R³ is H or methyl;

R⁴ is H or -L-Rx;

R⁵ is H, methyl, or phenyl;

R⁶ is H or bromine;

and R⁷ is methyl, phenyl, alkoxyphenyl, phenylethenyl, phenylbutatdienyl pyrrolyl, or thienyl;

where -L- is —

(CH₂)₂—

, —

(CH₂)₄—

, —

OCH₂C(O)NH(CH₂)₅—

, —

(CH₂)₂—

C(O)NH(CH₂)₅—

, —

(CH)₂C₆H₄OCH₂C(O)NH(CH₂)₅—

;

and Rx is a succinimidyl ester of a carboxylic acid; and

the specific binding pair member is an antibody or a streptavidin that contains a label that is an alkaline phosphatase and the fluorogenic substrate is a 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate, a 2-(5′

-chloro-2′

-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone, or ELF 39 reagent.