UniOne® TR-FRET His Check kit

The kit employs homogeneous time-resolved fluorescence (TR-FRET) technology for the detection of His-tagged proteins. It can be used to confirm whether the His-tag motif of a protein is recognizable by anti-His antibodies or to estimate the concentration of His-tagged proteins.

As illustrated, the His-tagged protein is labeled with an Acceptor (Ac-Protein His Tag), while the anti-His antibody is labeled with Europium (Eu-anti-His). Upon binding of the antibody to the His-tag on the protein, the proximity between the donor and acceptor enables fluorescence resonance energy transfer (FRET), resulting in a specific 665 nm emission signal from the acceptor. In the presence of other His-tagged proteins, they compete with Ac-Protein His Tag for binding to Eu-anti-His, thereby reducing FRET signal generation. Higher concentrations of competing His-tagged proteins lead to stronger competition and lower signals. The signal intensity is proportional to the binding between Ac-Protein His Tag and Eu-anti-His, with no washing steps required.