Rapid protein labeling and analysis
AssigneeLife Technologies Corporation
InventorsThomas Diller, George Hanson, Bethany Sutton, Timothy Updyke
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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.
Claims23
A method for protein analysis comprising fluorescently labeling a mixture of proteins, wherein said labeling comprises the steps of:
- a) forming a first reaction mixture by admixing the mixture of proteins with a composition comprising:
- i) a first buffer having a pH between pH 8 and pH 10, wherein said first buffer does not include buffer components or additives that have primary or secondary amine moieties;
- ii) an amine reactive fluorogenic reagent;
- and
- iii) acetone cyanohydrin, a nitrile, or an alkali cyanide,
- b) incubating the first reaction mixture for a first incubation time at a first temperature;
- c) forming a second reaction mixture by admixing a second buffer with the first reaction mixture, wherein the second buffer has a pH between pH 6 and pH 9;
- d) incubating for a second incubation time at a second temperature;
- and
- e) separating and visualizing the fluorescently labeled proteins.
Dependencies are inferred from the claim text.