Rapid protein labeling and analysis
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. A kit for fluorescently labeling a mixture of proteins comprising a tagged protein, said kit comprising:
a) a first labeling 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; and
a cyanohydrin or mandelonitrile,
wherein said first buffer does not include buffer components or additives that have primary or secondary amine moieties; and
b) a second labeling composition comprising a second buffer in the range from pH 6 to pH 10, and a tag binding fluorogenic reagent.
2. The kit of claim 1 , wherein the first labeling composition comprises an amine reactive fluorogenic reagent.
3. The kit of claim 2 , wherein said amine reactive fluorogenic reagent is 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde or 3-(2-furoyl)quinoline-2-carboxaldehyde).
4. The kit of claim 1 , wherein the first labeling 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.
5. The kit of claim 1 , wherein the second labeling composition further comprises a tag binding fluorogenic reagent that binds to at least four cysteine moieties, and at least one reducing agent, and wherein the amine reactive fluorogenic reagent or fluorescent dye of the first labeling composition has a first emission wavelength and the tag binding fluorogenic dye of the second labeling composition has a second emission wavelength, and the first emission wavelength and the second emission wavelength are different.
6. The kit of claim 5 , wherein the tag binding fluorogenic reagent is a biarsenical derivative of fluorescein or resorufin.
7. The kit of claim 1 , wherein said first labeling composition further comprises one or more of an anionic surfactant, a sugar or sugar alcohol, or an alkylating agent.
8. The kit of claim 1 , wherein said first labeling composition comprises acetone cyanohydrin.
9. The kit of claim 1 , wherein said first labeling composition comprises mandelonitrile.
10. The kit of claim 1 wherein said first labeling composition comprises acetone cyanohydrin and a mandelonitrile.
11. The kit of claim 1 , 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.
12. The kit of claim 1 , wherein said first buffer is a carbonate/bicarbonate buffer.