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Non-specific protein staining using merocyanine dyes

  • US 5,616,502 A
  • Filed: 05/19/1995
  • Issued: 04/01/1997
  • Est. Priority Date: 05/19/1995
  • Status: Expired due to Term
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3
Assignments
First Claim
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1. 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
    

    space="preserve" listing-type="equation">Q-B-M where Q is a nitrogen heterocycle of the formula ##STR59##  where R1, R2, R3 and R4 are optionally and independently H, Cl, F, C1 -C6 alkyl, C1 -C6 perfluoroalkyl, C1 -C6 alkoxy, amino, or amino substituted by 1-2 C1 -C6 alkyls;

    R5 and R6 are optionally and independently H, Cl, F, C1 -C6 alkyl, C1 -C6 perfluoroalkyl, C1 -C6 alkoxy, amino, or amino substituted by 1-2 C1 -C6 alkyls;

     or R5 and R6 taken in combination form a fused 6-membered aromatic ring that is optionally and independently substituted one or more times by Cl, F, C1 -C6 alkyl, C1 -C6 perfluoroalkyl, C1 -C6 alkoxy, amino, amino substituted by 1-2 C1 -C6 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, C1 -C6 alkyl, C1 -C6 perfluoroalkyl, C1 -C6 alkoxy, amino, amino substituted by 1-2 C1 -C6 alkyls;

    X is --S--, --O--, --NR7 --, or --CR7 R8 --, wherein R7 and R8 are optionally and independently H, Cl, F, phenyl, C1 -C6 alkyl.;

     or R7 and R8 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--NR9 --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 R9 that is a C2 -C6 alkyl that is optionally further substituted by hydroxy, carboxy, sulfo, amino, amino substituted by 1-2 C1 -C6 alkyls, or ammnonium substituted by 1-3 C1 -C6 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
    

    space="preserve" listing-type="equation">--(CR.sup.7 ═

    CR.sup.8).sub.n --where R7 and R8 are as defined previously;

    n=1, 2 or 3;

     and M is an electron pair-donating moiety of the formula ##STR60##  where R11 and R12 are independently H, F, Cl, or --CH3 ;

    R13 and R14 are independently C1 -C18 alkyl that is linear, branched, saturated or unsaturated, and is optionally substituted one or more times by F, hydroxy or C1 -C6 alkoxy;

     or R13 and R14 taken in combination form a 5- or 6-membered saturated ring containing 0 or 1 oxygen heteroatoms;

     or R13 taken in combination with R11 and R14 taken in combination with R12 independently are --(CH2)2 -- or --(CH2)3 --;

     or M is of the formula ##STR61## where Y is --OH, --SH, --O- or --S- ;

     Z is --OR15, --SR15, --N(R15)2 ;

    W is CN, --(C═

    O)--R16, or --(C═

    S)--R16 ;

    R15 is H or C1 -C6 alkyl;

    R16 is --OR15, --SR15, --N(R15)2 ;

     or M is of the formula ##STR62## where Y is as defined previously;

     Z'"'"' is --O--, --S--, or --NR17 --;

    W'"'"' is --O--, --S--, --NR17 --, --(C═

    O)--, --(C═

    S)--, or --(C═

    NR17)--;

    R17 is H, C1 -C6 alkyl, C1 -C6 carboxyalkyl, phenyl or phenyl substituted by sulfo;

    R18 is --O--, --S--, --NR17 --, --(C═

    O)--, --(C═

    S)--, or --(C═

    NR17)--;

    or W'"'"' and R18 taken in combination are --CR17 ═

    N--;

    or Z'"'"' and R18 taken in combination are --CR17 ═

    N--;

     or M is of the formula ##STR63## where Y and Z'"'"' are as defined previously;

     W" is --(C═

    O)--, --(C═

    S)--, or --(C═

    NR17)--;

    L is --(C═

    O)--, or --(C═

    S)--;

    R19 is --O--, --S--, --NR17 --;

    or W" and R19 taken in combination are --CR17 ═

    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.

      

    

  

  
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