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 - 34 . (canceled) 
     
     
         35 . A kit for fluorescently labeling a mixture of proteins comprising a tagged protein, said kit comprising:
 a) a first composition comprising a first buffer having a pH in the range from pH 8 to pH 10, an amine reactive fluorogenic reagent or an amine reactive fluorescent dye, a sugar or a sugar alcohol, and an anionic surfactant, wherein said first buffer does not include buffer components or additives that have primary or secondary amine moieties; and   b) a second composition comprising a second buffer in the range from pH 6 to pH 10, and optionally a tag binding fluorogenic reagent.   
     
     
         36 . The kit of  claim 35 , wherein the first composition comprises an amine reactive fluorogenic reagent and further comprises acetone cyanohydrin or mandelonitrile. 
     
     
         37 . The kit of  claim 36 , wherein said amine reactive fluorogenic reagent is 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde or 3-(2-furoyl)quinoline-2-carboxaldehyde). 
     
     
         38 . The kit of  claim 35 , wherein the first composition comprises an amine reactive fluorescent dye comprising 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. 
     
     
         39 . The kit of  claim 35 , wherein the second composition further comprises a tag binding fluorogenic reagent that binds to at least four cysteine moieties, and at least one reducing agent, wherein the amine reactive fluorogenic reagent or fluorescent dye has a first emission wavelength and the tag binding fluorogenic dye has a second emission wavelength, and the first emission wavelength and the second emission wavelength are different. 
     
     
         40 . The kit of  claim 39 , wherein the tag binding fluorogenic reagent is a biarsenical derivative of fluorescein or resorufin.