The polyhistidine tag (His tag) is the most prevalent affinity tag in recombinant protein research and the biotechnology field. Typically composed of six or more consecutive histidine residues fused to either the N-terminus or C-terminus of the target protein, it offers two core advantages: serving as a specific epitope for convenient detection of the target protein, and enabling protein affinity purification through coordination with metal ions. It is an essential tool throughout the entire process of recombinant protein expression and purification. In recombinant protein experiments and production, confirming the detectability of the His tag is a critical quality control prerequisite for the success of subsequent purification and functional assays. Current mainstream methods for detecting His tags are often characterized by lengthy workflows, cumbersome procedures, multiple variables, and low throughput, making it difficult to meet the demands of high-throughput, fast-paced R&D and production.
To address this core industry need, VKEY-BIO, leveraging its independently developed TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) technology platform, has launched the KeyTec® TR-FRET His Tag Check Kit. This kit is designed to confirm whether the His tag on a fusion protein can be recognized by an anti-His tag antibody, and can also be used to determine the relative concentration of His-tagged fusion proteins. Based on TR-FRET homogeneous assay technology and designed around a competitive immunoassay format, the entire process requires no washing steps, truly enabling a simplified "add-incubate-read" workflow. It is compatible with 96-well, 384-well, and 1536-well plates, making it suitable for all scenarios including basic research, high-throughput screening, and production quality control, effectively overcoming the pain points of lengthy procedures, cumbersome operations, and limited throughput associated with traditional methods.
This product is based on TR-FRET homogeneous assay technology with a competitive immunoassay design. An anti-His tag antibody conjugated to KeyTec® TR-FRET Solar Eu (fluorescence donor) specifically binds to the LA (fluorescence acceptor) conjugated His tag fusion protein. This brings the donor and acceptor into close proximity, enabling fluorescence resonance energy transfer upon excitation, generating a characteristic detection signal at 665 nm. His tags in the test sample competitively bind to the antibody; the higher the concentration of the His tag in the sample, the fewer donor-acceptor pairs capable of efficient energy transfer are formed, resulting in a decrease in the detection signal at 665 nm.
Figure 1: Schematic diagram of the KeyTec® TR-FRET His tag detection principle
Detection using a recombinant protein standard containing a polyhistidine tag shows low background signal and an excellent assay window (S/B > 27).
Assay results demonstrate that the kit can recognize and capture His tags fused at the N-terminus or C-terminus of proteins and exposed on the protein surface, exhibiting excellent response performance to His tags of varying lengths.
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