The HiBiT assay represents a significant advancement in molecular biology, offering a sophisticated method for understanding proteins within living systems. This technique allows researchers to detect and quantify proteins with high sensitivity, providing insights into their abundance and behavior. It operates on the principle of bioluminescence, where light is produced through a biochemical reaction, enabling precise measurements of target proteins. The HiBiT system offers a streamlined approach, reducing the need for traditional, more laborious detection methods.
The HiBiT System’s Core Components
The HiBiT system relies on the interplay of three distinct components to generate its signal. The first component is the HiBiT tag, a peptide of just 11 amino acids. This tag is engineered to be attached to a specific target protein that researchers wish to study, effectively labeling it for detection. The small size of the HiBiT tag minimizes its potential to interfere with the natural function of the fused protein.
The second component is LgBiT, which is a larger fragment of an enzyme called NanoLuc luciferase, comprising 156 amino acids. On its own, LgBiT is inactive and cannot produce light. However, it possesses a strong affinity for the HiBiT tag. The third component is furimazine, a specialized substrate. Furimazine is the molecule that will react with the reconstituted enzyme to generate the detectable light signal.
Illuminating Proteins: How HiBiT Works
The operational mechanism of the HiBiT assay centers on a concept known as “complementation.” When the HiBiT tag, fused to a target protein, encounters the LgBiT fragment, they rapidly associate. This association is driven by their high affinity for each other, effectively reassembling a complete and functional NanoLuc luciferase enzyme.
Once the full NanoLuc enzyme is reconstituted, it becomes biochemically active. This active enzyme then catalyzes a reaction with the furimazine substrate. This reaction converts furimazine, producing a bright, measurable light signal. The intensity of this emitted light is directly proportional to the amount of functional NanoLuc enzyme present, which in turn correlates with the quantity of the HiBiT-tagged target protein in the sample.
This direct relationship between light output and protein amount makes the HiBiT assay a highly quantitative method. Researchers can use a luminometer to measure the light, providing a sensitive and accurate readout of protein levels. The simplicity of adding a single reagent containing LgBiT and furimazine, followed by light measurement, streamlines the detection process.
Broadening Research Horizons
The HiBiT assay has expanded the capabilities of biological research across various applications. It offers a precise method for quantifying protein levels, allowing scientists to monitor changes in protein abundance under different experimental conditions. For instance, it can be used to study protein degradation pathways, even in response to specific inhibitors.
The system is also valuable for analyzing protein-protein interactions, as the light signal can indicate when two proteins, one tagged with HiBiT and another associated with LgBiT, come together. Researchers can also use HiBiT to track the localization of proteins within cells. The technology offers high sensitivity, enabling the detection of proteins even at low, endogenous expression levels.
A key application of HiBiT involves its integration with genome editing technologies like CRISPR/Cas9. This combination allows scientists to directly insert the HiBiT tag into the cell’s own DNA at the gene’s location. This means the protein is tagged at its natural physiological levels, rather than being artificially overexpressed, which can sometimes alter protein behavior.
By tagging endogenous proteins, researchers can study them in their native cellular environment, providing more accurate and biologically relevant data. This approach removes potential artifacts that might arise from traditional overexpression methods. The small size of the HiBiT tag makes it an efficient choice for CRISPR-mediated genome editing, simplifying the process of creating tagged cell lines for various studies.