Rapid protein labeling and analysis

Abstract

The present invention provides methods and compositions for labeling, separating and analyzing proteins, particularly a specific protein of interest within a cell lysate or in a mixture of proteins. The proteins are labeled with an amine reactive or thiol reactive fluorescent dye, or an amine reactive fluorogenic reagent that becomes fluorescent upon reacting to amine groups located on the protein. Following the labeling step, the proteins within the mixture can be separated and analyzed. In a further embodiment, a tag binding fluorogenic reagent that can bind to a tag on a tagged protein is added to specifically label the protein of interest.

1 - 40 . (canceled) 
     
     
         41 . A kit for fluorescently labeling a mixture of proteins comprising:
 a) a labeling composition comprising:
 a first buffer having a pH in the range from pH 8 to pH 10, wherein said first buffer lacks components or additives that have primary or secondary amine moieties; 
 an amine reactive fluorogenic reagent; 
 a cyanohydrin or mandelonitrile; 
 a second buffer in the range from pH 6 to pH 10; and, optionally; 
   b) a tag binding fluorogenic reagent.   
     
     
         42 . The kit of  claim 41 , wherein said amine reactive fluorogenic reagent is an amine reactive fluorescent dye. 
     
     
         43 . The kit of  claim 41 , wherein said amine reactive fluorogenic reagent is 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde or 3-(2-furoyl)quinoline-2-carboxaldehyde). 
     
     
         44 . The kit of  claim 41 , wherein the amine reactive fluorescent dye comprises a fluorescent moiety selected from the group consisting of a fluorescein moiety, a rhodamine moiety, an acridine moiety, a coumarin moiety, an indole moiety, an isoindole moiety, an indolizine moiety, a quinoline moiety, an isoquinoline moiety, a chromene moiety, a xanthene moiety, a naphthalene moiety, a pyrene moiety, a bimane moiety and a BODIPY moiety. 
     
     
         45 . The kit of  claim 41 , wherein the tag binding fluorogenic reagent binds to at least four cysteine moieties. 
     
     
         46 . The kit of  claim 41 , wherein the optional tag binding fluorogenic reagent is a biarsenical derivative of fluorescein or resorufin. 
     
     
         47 . The kit of  claim 41 , further comprising one or more of an anionic surfactant, a sugar or sugar alcohol, or an alkylating agent. 
     
     
         48 . The kit of  claim 41 , wherein said cyanohydrin is acetone cyanohydrin. 
     
     
         49 . The kit of  claim 41 , wherein said first buffer is selected from the list consisting of carbonate/bicarbonate buffer, phosphate buffer, borate buffer, and N,N-Bis(2-hydroxyethyl)glycine (Bicine) buffer. 
     
     
         50 . The kit of  claim 41 , wherein said first buffer is a carbonate/bicarbonate buffer. 
     
     
         51 . The kit of  claim 41 , further comprising at least one reducing agent. 
     
     
         52 . The kit of  claim 41 , wherein the amine reactive fluorogenic reagent has a first emission wavelength, the optional tag binding fluorogenic dye has a second emission wavelength, the first emission wavelength and the second emission wavelength being different.