Direct detection of histidine tagged biomolecules on electrophoretic gel

Abstract

A method is disclosed for the detection of a His-tagged target biomolecule while said molecule remains in the gel in which it has been separated from other constituents by electrophoresis. The method involves, while the separated His-tagged biomolecule remains in the gel, forming a chelate between the His-tagged molecule, a lanthanide metal ion which exhibits fluorescence, and a sensitizer which enhances the fluorescence of the ion. The gel is then exposed to a u.v. light source to thereby generate a fluorogenic signal indicating the presence of the chelate and, in turn, the His-tagged biomolecule. The fluorogenic signal can then be detected either by direct visualization and/or by other means, such as photographically, e.g., film or charged coupled device (CCD) imaging.

What is claimed is:  
     
         1 . A method for the direct detection of a histidine tagged biomolecule on an electrophoretic gel comprising (1) separating the biomolecule from other substances by electrophoresis on a gel, (2) while said molecule remains so separated, forming a chelate between said molecule, a lanthanide metal ion which exhibits fluorescence, and a sensitizer which enhances the fluorescence of the ion, and (3) exposing the gel containing said chelate to a u.v. light source thereby generating a fluorescent signal indicative of the presence of the chelate and, in turn, the histidine tagged biomolecule, and (3) detecting the signal so generated.  
     
     
         2 . The method of  claim 1  wherein the lanthanide metal ion is terbium or europium and the sensitizer is a dipicolinic acid.  
     
     
         3 . The method of  claim 2  wherein the lanthanide metal ion is terbium.  
     
     
         4 . The method of  claim 2  or  3  wherein the dipicolinic acid is pyridine, 2,6 dicarboylic acid.  
     
     
         5 . The method of  claim 1  wherein said chelate of the histidine tagged biomolecule, the lanthanide metal ion and the dipicolinic acid is formed by first chelating the histidine tagged biomolecule with the lanthanide metal ion followed by chelation with the sensitizer.  
     
     
         6 . The method of  claim 5  wherein the lanthanide metal ion is terbium or europium and the sensitizer is a dipicolinic acid.  
     
     
         7 . The method of  claim 6  wherein the lanthanide metal ion is terbium.  
     
     
         8 . The method of  claim 6  or  7  wherein the dipicolinic acid is pyridine, 2,6 dicarboylic acid.  
     
     
         9 . A kit for the direct detection of histidine tagged biomolecules on an electrophoretic gel, said kit containing two separately packaged components and a written instruction sheet, one of said components being an aqueous solution of a lanthanide metal ion which exhibits fluorescence and the other of said components being an aqueous solution of a sensitizer which enhances the fluorescence of the ion.  
     
     
         10 . The kit of  claim 9  wherein the lanthanide metal ion is terbium.  
     
     
         11 . The kit of  claim 10  wherein the sensitizer is pyridine, 2,6 dicarboxylic acid.