Fluorescent dyes, fluorescent dye kits, and methods of preparing labeled molecules

Abstract

The present invention provides methods, compositions, and kits useful in preparing labeled molecules, which are useful in the detection of binding partners.

What is claimed is: 
     
         1 .- 104 . (canceled) 
     
     
         105 . A method for staining one or more biological targets comprising:
 (a) preparing one or more labeled proteins by:
 (i) providing an amine-reactive detectable dye or an amine-reactive detectable label which is a biotin or a digoxin and a first amine which is a weak primary aliphatic amine; 
 (ii) combining the amine-reactive detectable label or the amine-reactive detectable dye and the first amine, in a buffer, with a sample solution comprising a target protein, the target protein comprising a second amine, to form a combined solution; and 
 (iii) allowing the amine reactive detectable dye or the amine amine-reactive detectable label to react with the first amine and the second amine in said combined solution; wherein said amine-reactive detectable dye or amine-reactive detectable label comprises an activated ester and wherein the amine-reactive detectable label or amine-reactive detectable dye in said combined solution reacts more strongly with the second amine than the first amine, thereby producing the target protein labeled with said target protein labeled with said detectable or said detectable label; 
   (b) exposing said one or more biological targets to said one or more labeled proteins, such that said labeled protein binds to said one or more biological targets thereby staining said one or more biological targets.   
     
     
         106 . The method of  claim 105 , wherein step (b) is performed without purification of said one or more labeled proteins from step (a). 
     
     
         107 . The method of  claim 105 , with the proviso that a quencher is not added in step (a). 
     
     
         108 . The method of  claim 105 , wherein said staining takes place in vivo. 
     
     
         109 . The method of  claim 105 , wherein said staining takes place in vitro. 
     
     
         110 . The method of  claim 105 , wherein said biological target is a cell surface protein. 
     
     
         111 . The method of  claim 105 , wherein said biological target is an intracellular protein. 
     
     
         112 . The method of  claim 105 , wherein two or more labeled proteins are present, and each of said two or more labeled proteins bind to one or more biological targets. 
     
     
         113 . The method of  claim 112 , wherein each of said two or more labeled proteins comprise different dyes, such that said exposing step (b) renders said different binding partners optically distinguishable. 
     
     
         114 . The method of  claim 105 , further comprising analyzing stained biological targets by flow cytometry, western blot, or by microscopy. 
     
     
         115 . The method of  claim 105 , further comprising sorting cells stained by said exposing using a fluorescence activated cell sorter. 
     
     
         116 . The method of  claim 105 , further comprising visualizing fluorescence of the stained one or more biological targets. 
     
     
         117 . The method of  claim 105 , further comprising diagnosing a condition of a subject based on analysis of said stained one or more biological targets. 
     
     
         118 . A kit for preparing a labeled protein comprising:
 (a) an amine-reactive detectable dye or an amine-reactive detectable label which is biotin or a digoxin; and a first amine which is a weak primary aliphatic amine;   (b) instructions directing a user to combine, in a buffer, the amine-reactive detectable label, the first amine, and a sample solution comprising a target protein, the target protein comprising a second amine, to form a combined solution; and   wherein said amine-reactive detectable dye or amine-reactive detectable label comprises an activated ester and wherein the amine-reactive detectable label or amine-reactive detectable dye in said combined solution reacts more strongly with the second amine than the first amine, thereby producing the target protein labeled with said target protein labeled with said detectable or said detectable label.   
     
     
         119 . The kit of  claim 118 , wherein said buffer is selected from the group consisting of: a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, or a combination thereof. 
     
     
         120 . The kit of  claim 118 , wherein said buffer is alkaline. 
     
     
         121 . The kit of  claim 118 , wherein the pH of the buffer is in the range from 7 to 10. 
     
     
         122 . The kit of  claim 118 , wherein said amine is tris(hydroxymethyl)aminomethane (Tris). 
     
     
         123 . The kit of  claim 122 , wherein said tris(hydroxymethyl)aminomethane is present in said buffer in a concentration greater than 20 mM. 
     
     
         124 . The kit of  claim 118 , wherein said activated ester is N-hydroxy succinimidyl ester, N-hydroxy sulfosuccinimidyl ester, p-sulfo-tetrafluorophenol ester, or a combination thereof. 
     
     
         125 . The kit of  claim 118 , wherein said amine-reactive dye or amine reactive-label comprises one or more sulfonate groups. 
     
     
         126 . The kit of  claim 118 , wherein said amine-reactive dye or amine reactive-label comprises one or more water-soluble polymer groups. 
     
     
         127 . The kit of  claim 126 , wherein said water-soluble polymer group is a polyethylene glycol.