Hexahistidine ((His)6) tags are used to purify genetically engineered proteins. Herein, we describe the construction of a 'turn-on' fluorescent probe system that consists of the fluorescence quencher, Dabcyl, conjugated to (His)6, and fluorescent tetramethylrhodamine conjugated to nitrilotriacetic acid, which, in the presence of Ni2+, can bind (His)6. The system is turned off when Dabcyl-(His)6 is bound to the fluorescent nitrilotriacetic acid derivative. The binding strength of this system was assessed using electrospray ionization mass spectrometry, fluorescence correlation spectroscopy, and fluorescence intensity distribution analysis-polarization. Although there was no significant enhancement in fluorescence after addition of an equimolar amount of ubiquitin, the fluorescence increased from 14% to 40% of its initial intensity when an equimolar amount of (His)6-ubiquitin was added. Therefore, this system should be able to specifically recognize (His)6-proteins with good resolution and has the additional advantage that a washing step is not required to remove fluorescent probe, that is, not bound to the (His) 6-protein.
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