LABELING OF IMMOBILIZED PROTEINS USING DIPYRROMETHENEBORON DIFLUORIDE DYES
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.
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:
wherein each of R 1 through R 7 are independently selected from the group consisting of H, halogen, -L-Rx, and substituted or unsubstituted C 1 -C 6 alkyl, aryl, arylethenyl, arylbutadienyl, and heteroaryl;
provided that one or more of R 1 through R 7 is H,
two or more of R 1 through R 7 is nonhydrogen, and
only one of R 1 through R 7 is -L-Rx, where L is a spacer having 1-24 nonhydrogen atoms and Rx is a maleimide or a succinimidyl ester of a carboxylic acid;
such that the dipyrrometheneboron difluoride dye has an absorption maximum between 495 nm and 640 nm;
b. a specific binding pair member that contains a label and that selectively binds to a target that is its complementary binding pair.
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 R 1 is methyl or -L-Rx; R 2 is H, bromine, or -L-Rx; R 3 is H or methyl; R 4 is H or -L-Rx; R 5 is H, methyl, or phenyl; R 6 is H or bromine;
and R 7 is methyl, phenyl, alkoxyphenyl, phenylethenyl, phenylbutatdienyl pyrrolyl, or thienyl;
where -L- is —(CH 2 ) 2 —, —(CH 2 ) 4 —, —OCH 2 C(O)NH(CH 2 ) 5 —, —(CH 2 ) 2 —C(O)NH(CH 2 ) 5 —, —(CH) 2 C 6 H 4 OCH 2 C(O)NH(CH 2 ) 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.