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US 5,616,502 AGranted
Non-specific protein staining using merocyanine dyes
AssigneeMolecular Probes, Inc.
InventorsRichard P. Haugland, Victoria L. Singer, Laurie J. Jones, Thomas H. Steinberg
Life of this patent
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Abstract
The present invention describes the use of a variety of merocyanine dyes and substituted merocyanine dyes for detecting and quantifying poly(amino acids), including peptides, polypeptides and proteins. The labeled proteins or peptides are highly colored, but are also detected by their strong fluorescence enhancement. Poly(amino acids) are detected in solution, in electrophoretic gels, and on solid supports, including blots and dipsticks. The present method of staining is highly sensitive, extremely facile, and relatively non-selective.
Claims38
A method of detecting a poly(amino acid), comprising the steps of:
- a) combining a sample mixture that is thought to contain a poly(amino acid) with a staining mixture that contains one or more merocyanine dyes to form a combined mixture;
- wherein each merocyanine dye independently has the formula
- Q-B-M
- where Q is a nitrogen heterocycle of the formula ##STR59## where R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are optionally and independently H, Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, C.sub.1 -C.sub.6 alkoxy, amino, or amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls:
- R.sup.5 and R.sup.6 are optionally and independently H, Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, C.sub.1 -C.sub.6 alkoxy, amino, or amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls:
- or R.sup.5 and R.sup.6 taken in combination form a fused 6-membered aromatic ring that is optionally and independently substituted one or more times by Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, C.sub.1 -C.sub.6 alkoxy, amino, amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls, or said fused 6-membered aromatic ring is optionally substituted by an additional fused 6-membered aromatic ring that is optionally and independently substituted by Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, C.sub.1 -C.sub.6 alkoxy, amino, amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls;
- X is --S--, --O--, --NR.sup.7 --, or --CR.sup.7 R.sup.8 --, wherein R.sup.7 and R.sup.8 are optionally and independently H, Cl, F, phenyl, C.sub.1 -C.sub.6 alkyl.;
- or R.sup.7 and R.sup.8 taken in combination complete a 5- or 6-membered saturated fine:
- TAIL is attached to Q through a carbon atom and contains 1-22 non-hydrogen atoms, wherein said non-hydrogen atoms are selected from the group consisting of C, O, N and S, such that each heteroatom is separated from any adjacent heteroatoms by at least two carbon atoms:
- and further such that TAIL is composed of carbon-carbon bonds (C--C), ether bonds (C--O--C), thioether bonds (C--S--C) or amine bonds (C--NR.sup.9 --C);
- where any carbon atom in TAIL is optionally further substituted by hydroxy, carboxy, sulfo, amino or ammonium;
- and where any amine bond, amino or ammonium in TAIL is optionally substituted by an R.sup.9 that is a C.sub.2 -C.sub.6 alkyl that is optionally further substituted by hydroxy, carboxy, sulfo, amino, amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls, or ammnonium substituted by 1-3 C.sub.1 -C.sub.6 alkyls, or said N atoms form either one or two saturated 5- or 6-membered rings in combination with additional C or N atoms in TAIL;
- B is a covalent bridge having the formula
- --(CR.sup.7 .dbd.CR.sup.8).sub.n --
- where R.sup.7 and R.sup.8 are as defined previously;
- n=1, 2 or 3;
- and M is an electron pair-donating moiety of the formula ##STR60## where R.sup.11 and R.sup.12 are independently H, F, Cl, or --CH.sub.3 ;
- R.sup.13 and R.sup.14 are independently C.sub.1 -C.sub.18 alkyl that is linear, branched, saturated or unsaturated, and is optionally substituted one or more times by F, hydroxy or C.sub.1 -C.sub.6 alkoxy;
- or R.sup.13 and R.sup.14 taken in combination form a 5- or 6-membered saturated ring containing 0 or 1 oxygen heteroatoms;
- or R.sup.13 taken in combination with R.sup.11 and R.sup.14 taken in combination with R.sup.12 independently are --(CH.sub.2).sub.2 -- or --(CH.sub.2).sub.3 --;
- or M is of the formula ##STR61## where Y is --OH, --SH, --O.sup.- or --S.sup.- ;
- Z is --OR.sup.15, --SR.sup.15, --N(R.sup.15).sup.2 ;
- W is CN, --(C.dbd.O)--R.sup.16, or --(C.dbd.S)--R.sup.16 ;
- R.sup.15 is H or C.sub.1 -C.sub.6 alkyl;
- R.sup.16 is --OR.sup.15, --SR.sup.15, --N(R.sup.15).sub.2 ;
- or M is of the formula ##STR62## where Y is as defined previously;
- Z' is --O--, --S--, or --NR.sup.17 --;
- W' is --O--, --S--, --NR.sup.17 --, --(C.dbd.O)--, --(C.dbd.S)--, or --(C.dbd.NR.sup.17)--;
- R.sup.17 is H, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 carboxyalkyl, phenyl or phenyl substituted by sulfo;
- R.sup.18 is --O--, --S--, --NR.sup.17 --, --(C.dbd.O)--, --(C.dbd.S)--, or --(C.dbd.NR.sup.17)--;
- or W' and R.sup.18 taken in combination are --CR.sup.17 .dbd.N--;
- or Z' and R.sup.18 taken in combination are --CR.sup.17 .dbd.N--;
- or M is of the formula ##STR63## where Y and Z' are as defined previously;
- W" is --(C.dbd.O)--, --(C.dbd.S)--, or --(C.dbd.NR.sup.17)--;
- L is --(C.dbd.O)--, or --(C.dbd.S)--;
- R.sup.19 is --O--, --S--, --NR.sup.17 --;
- or W" and R.sup.19 taken in combination are --CR.sup.17 .dbd.N--;
- such that the resulting heterocycle does not include any O--O, S--S, O--S, or N--N--N bonds;
- b) heating tile sample mixture prior to combining with the staining mixture, or heating the combined mixture:
- c) incubating the combined mixture for a time sufficient for the dye in the staining mixture to associate with the poly(amino acid) in the sample mixture to form a dye-poly(amino acid) complex that gives a detectable optical response upon illumination;
- d) illuminating said dye-poly(amino acid) complex;
- and
- e) observing said detectable optical response.
40 more elements
A combination consisting essentially of:
- a) one or more styryl dyes having the formula
- Q-B-M
- where Q is a nitrogen heterocycle of the formula ##STR64## where R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are optionally and independently H, Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl,
- C.sub.1 -C.sub.6 alkoxy, amino, or amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls;
- R.sup.5 and R.sup.6 are optionally and independently H, Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, C.sub.1 -C.sub.6 alkoxy, amino, or amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls;
- or R.sup.5 and R.sup.6 taken in combination form a fused 6-membered aromatic ring that is optionally and independently substituted one or more times by Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, C.sub.1 -C.sub.6 alkoxy, amino, amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls, or said fused 6-membered aromatic ring is optionally substituted by an additional fused 6-membered aromatic ring that is optionally and independently substituted by Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, C.sub.1 -C.sub.6 alkoxy, amino, amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls;
- X is --S--, --O--, --NR.sup.7 --, or --CR.sup.7 R.sup.8 --, wherein R.sup.7 and R.sup.8 are optionally and independently H, Cl, F, phenyl, C.sub.1 -C.sub.6 alkyl;
- or R.sup.7 and R.sup.8 taken in combination complete a 5- or 6-membered saturated ring;
- TAIL is attached to Q through a carbon atom and contains 1-22 non-hydrogen atoms, wherein said non-hydrogen atoms are selected from the group consisting of C, O, N and S, such that each heteroatom is separated from any adjacent heteroatoms by at least two carbon atoms;
- and further such that TAIL is composed of carbon-carbon bonds (C--C), ether bonds (C--O--C), thioether bonds (C--S--C) or amine bonds (C--NR.sup.9 --C);
- where any carbon atom in TAIL is optionally further substituted by hydroxy, carboxy, sulfo, amino or ammonium;
- and where any amine bond, amino or ammonium in TAIL is optionally substituted by an R.sup.9 that is a C.sub.2 -C.sub.6 alkyl that is optionally further substituted by hydroxy, carboxy, sulfo, amino, amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls, or ammonium substituted by 1-3 C.sub.1 -C.sub.6 alkyls, or said N atoms form either one or two saturated 5- or 6-membered rings in combination with additional C or N atoms in TAIL;
- B is a covalent bridge having tile formula
- --(CR.sup.7 .dbd.CR.sup.8).sub.n --
- where R.sup.7 and R.sup.8 are as defined previously;
- n=1, 2 or 3;
- and M is an electron pair-donating moiety of the formula ##STR65## where R.sup.11 and R.sup.12 are independently H, F, Cl, or --CH.sub.3 ;
- R.sup.13 and R.sup.14 are independently C.sub.1 -C.sub.18 alkyl that is linear, branched, saturated or unsaturated, and is optionally substituted one or more times by F, hydroxy or C.sub.1 -C.sub.6 alkoxy;
- or R.sup.13 and R.sup.14 taken in combination form a 5- or 6-membered saturated ring containing 0 or 1 oxygen heteroatoms;
- or R.sup.13 taken in combination with R.sup.11 and R.sup.14 taken in combination with R.sup.12 independently are --(CH.sub.2).sub.2 -- or --(CH.sub.2).sub.3 --;
- b) a detergent;
- and
- c) a poly(amino acid),
- in a cell-free aqueous solution where said detergent is present at a concentration less than the critical micelle concentration for that detergent.
21 more elements
A kit for staining the poly(amino acids) in a sample, comprising:
- a) one or more merocyanine dyes, wherein each merocyanine dye independently has the formula
- Q-B-M
- where Q is a nitrogen heterocycle of the formula ##STR66## where R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are optionally and independently H, Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, C.sub.1 -C.sub.6 alkoxy, amino or amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls:
- R.sup.5 and R.sup.6 are optionally and independently H, Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, C.sub.1 -C.sub.6 alkoxy, amino, or amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls;
- or R.sup.5 and R.sup.6 taken in combination form a fused 6-membered aromatic ring that is optionally and independently substituted one or more times by Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, C.sub.1 -C.sub.6 alkoxy, amino, amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls, or said fused 6-membered aromatic ring is optionally substituted by an additional fused 6-membered aromatic ring that is optionally and independently substituted by Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, C.sub.1 -C.sub.6 alkoxy, amino, amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls;
- X is --S--, --O--, --NR.sup.7 --, or --CR.sup.7 R.sup.8 --, wherein R.sup.7 and R.sup.8 are optionally and independently H, Cl, F, phenyl, C.sub.1 -C.sub.6 alkyl,;
- or R.sup.7 and R.sup.8 taken in combination complete a 5- or 6-membered saturated ring;
- TAIL is attached to Q through a carbon atom and contains 1-22 non-hydrogen atoms, wherein said non-hydrogen atoms are selected from the group consisting of C, O, N and S, such that each heteroatom is separated from any adjacent heteroatoms by at least two carbon atoms;
- and further such that TAIL is composed of carbon-carbon bonds (C--C), ether bonds (C--O--C), thioether bonds (C--S--C) or amine bonds (C--NR.sup.9 --C):
- where any carbon atom in TAIL is optionally further substituted by hydroxy, carboxy, sulfo, amino or ammonium;
- and where any amine bond, amino or ammonium in TAIL is optionally substituted by an R.sup.9 that is a C.sub.2 -C.sub.6 alkyl that is optionally further substituted by hydroxy, carboxy, sulfo, amino, amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls, or ammonium substituted by 1-3 C.sub.1 -C.sub.6 alkyls, or said N atoms form either one or two saturated 5- or 6-membered rings in combination with additional C or N atoms in TAIL;
- B is a covalent bridge having the formula
- --(CR.sup.7 .dbd.CR.sup.8).sub.n --
- where R.sup.7 and R.sup.8 are as defined previously;
- n=1, 2 or 3;
- and M is an electron pair-donating moiety of the formula ##STR67## where R.sup.11 and R.sup.12 are independently H, F, Cl, or --CH.sub.3 ;
- R.sup.13 and R.sup.14 are independently C.sub.1 -C.sub.18 alkyl that is linear, branched, saturated or unsaturated, and is optionally substituted one or more times by F, hydroxy or C.sub.1 -C.sub.6 alkoxy;
- or R.sup.13 and R.sup.14 taken in combination form a 5- or 6-membered saturated ring containing 0 or 1 oxygen heteroatoms;
- or R.sup.13 taken in combination with R.sup.11 and R.sup.14 taken in combination with R.sup.12 independently are --(CH.sub.2).sub.2 -- or --(CH.sub.2).sub.3 --;
- or M is of the formula ##STR68## where Y is --OH, --SH, --O.sup.- or --S.sup.- ;
- Z is --OR.sup.15, --SR.sup.15, --N(R.sup.15).sub.2 ;
- W iS CN, --(C.dbd.O)--R.sup.16, or --(C.dbd.S)--R.sup.16 ;
- R.sup.15 is H or C.sub.1 -C.sub.6 alkyl:
- R.sup.16 is --OR.sup.15, --SR.sup.15, --N(R.sup.15).sub.2 ;
- or M is of the formula ##STR69## where Y is as defined previously:
- Z' is --O--, --S--, or --NR.sup.17 --;
- W' is --O--, --S--, --NR.sup.17, --(C.dbd.O)--, --(C.dbd.S)--, or --(C.dbd.NR.sup.17)--;
- R.sup.17 is H, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 carboxyalkyl, phenyl or phenyl substituted by sulfo;
- R.sup.18 is --O--, --S--, --NR.sup.17 --, --(C.dbd.O)--, --(C.dbd.S)--, or --(C.dbd.NR.sup.17)--;
- or W' and R.sup.18 taken in combination are --CR.sup.17 .dbd.N--;
- or Z' and R.sup.18 taken in combination are --CR.sup.17 .dbd.N--;
- or M is of the formula ##STR70## where Y and Z' are as defined previously:
- W" is --(C.dbd.O)--, --(C.dbd.S)--, or --(C.dbd.NR.sup.17)--;
- L is --(C.dbd.O)--, or --(C.dbd.S)--;
- R.sup.19 is --O--, --S--, or --NR.sup.17 --;
- or W" and R.sup.19 taken in combination are --CR.sup.17 .dbd.N--;
- such that the resulting heterocycle does not include any O--O, S--S, O--S, or N--N--N bonds;
- and
- b) instructions for combining said merocyanine dye or dyes with a sample containing or thought to contain poly(amino acids),;
- said instructions comprising
- i) combining a sample that is thought to contain a poly(amino acid) with a staining mixture that contains said merocyanine dye or dyes to form a combined mixture;
- ii) incubating the combined mixture for a time sufficient for the dye in the staining mixture to associate with the poly(amino acid) in the sample mixture to form a dye-poly(amino acid) complex that gives a detectable optical response upon illumination.
39 more elements
A method of detecting a poly(amino acid), comprising the steps of:
- a) combining a sample mixture that is thought to contain a poly(amino acid) with a staining mixture that contains one or more styryl dyes having the formula
- Q-B-M
- where Q is a nitrogen heterocycle of the formula ##STR73## where R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are optionally and independently H, Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, C.sub.1 -C.sub.6 alkoxy, amino, or amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls;
- R.sup.5 and R.sup.6 are optionally and independently H, Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, C.sub.1 -C.sub.6 alkoxy, amino, or amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls;
- or R.sup.5 and R.sup.6 taken in combination form a fused 6-membered aromatic ring that is optionally and independently substituted one or more times by Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, C.sub.1 -C.sub.6 alkoxy, amino, amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls, or said fused 6-membered aromatic ring is optionally substituted by an additional fused 6-membered aromatic ring that is optionally and independently substituted by Cl, F, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, C.sub.1 -C.sub.6 alkoxy, amino, amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls;
- X is --S--, --O--, --NR.sup.7 --, or --CR.sup.7 R.sup.8 --, wherein R.sup.7 and R.sup.8 are optionally and independently H, Cl, F, phenyl, C.sub.1 -C.sub.6 alkyl;
- or R.sup.7 and R.sup.8 taken in combination complete a 5- or 6-membered saturated ring;
- TAIL is attached to Q through a carbon atom and contains 1-22 non-hydrogen atoms, wherein said non-hydrogen atoms are selected from the group consisting of C, O, N and S, such that each heteroatom is separated from any adjacent heteroatoms by at least two carbon atoms;
- and further such that TAIL is composed of carbon-carbon bonds (C--C), ether bonds (C--O--C), thioether bonds (C--S--C) or amine bonds (C--NR.sup.9 --C);
- where any carbon atom in TAIL is optionally further substituted by hydroxy, carboxy, sulfo, amino or ammonium;
- and where any amine bond, amino or ammonium in TAIL is optionally substituted by an R.sup.9 that is a C.sub.2 -C.sub.6 alkyl that is optionally further substituted by hydroxy, carboxy, sulfo, amino, amino substituted by 1-2 C.sub.1 -C.sub.6 alkyls, or ammonium substituted by 1-3 C.sub.1 -C.sub.6 alkyls, or said N atoms form either one or two saturated 5- or 6-membered rings in combination with additional C or N atoms in TAIL;
- B is a covalent bridge having the formula
- --(CR.sup.7 .dbd.CR.sup.8).sub.n --
- where R.sup.7 and R.sup.8 are as defined previously;
- n=1, 2 or 3;
- and M is an electron pair-donating moiety of the formula ##STR74## where R.sup.11 and R.sup.12 are independently H, F, Cl, or --CH.sub.3 ;
- R.sup.13 and R.sup.14 are independently C.sub.1 -C.sub.18 alkyl that is linear, branched, saturated or unsaturated, and is optionally substituted one or more times by F, hydroxy or C.sub.1 -C.sub.6 alkoxy;
- or R.sup.13 and R.sup.14 taken in combination form a 5- or 6-membered saturated ring containing 0 or 1 oxygen heteroatoms;
- or R.sup.13 taken in combination with R.sup.11 and R.sup.14 taken in combination with R.sup.12 independently are --(CH.sub.2).sub.2 -- or --(CH.sub.2).sub.3 --;
- to form a combined mixture;
- b) incubating the combined mixture for a time sufficient for the dye in the staining mixture to associate with the poly(amino acid) in the sample mixture to form a dye-poly(amino acid) complex that gives a detectable optical response upon illumination;
- c) illuminating said dye-poly(amino acid) complex;
- and
- d) observing said detectable optical response.
21 more elements
Dependencies are inferred from the claim text.