Issue link: https://resources.nanotempertech.com/i/1533227
16 Spectral Shift Spectral Shi is an immobilization-free technology to quantify molecular interactions between a fluorescently labeled target molecule (target) and an unlabeled binding partner (ligand). As it is a rapid, isothermal, non-destructive measurement, it permits sample re-analysis, temperature-sensitive studies and studies of fragile molecules that destabilize quickly. The highly sensitive and robust detection allows researchers to work with small amounts of labeled target and even suboptimal samples that contain aggregation or precipitation. Conclusion Spectral Shi is the go-to solution for precise measurements of biomolecular interactions free in solution. Spectral Shi technology excels at interaction characterization of a wide range of biomolecules due to its high sensitivity. Its immobilization-free setup, minimal sample consumption, and flexibility across buffers and molecule types also makes it a great fit for labs that have a variety of projects or work with challenging samples. How it works Spectral Shift measures the fluorescence intensity of a labeled protein mixed with a dilution series of an unlabeled ligand. Highly precise optics monitor subtle shifts in the emission wavelength down to picometer resolution—hence the name Spectral Shift. These shifts occur largely due to changes in the polarity of the fluorophore's molecular environment. To analyze the spectral shift, fluorescence is recorded at 650 and 670 nm, and then the ratiometric measurement of 670/650 nm is plotted versus the ligand concentration, canceling out pipetting errors. The affinity constant (K d ) is automatically determined at the end of each run without any additional or lengthy data analysis. Strengths Like TRIC, Spectral Shift measurements are performed with microliter sample volumes and in solution, allowing for close-to-native conditions. Measurements can be run in all sorts of buffers including things like lysates and serum, with molecules of all weights and classes, and are completed in minutes, which makes Spectral Shi suitable for basically any research project. The major additional strength of Spectral Shi is its increased sensitivity and, consequently, its reliability: the way the signal is detected produces results with a very high signal-to-noise ratio, allowing to find binding partners your primary assay missed, or to identify false positives. This is especially useful for interactions that are difficult to characterize with other methods. Weaknesses Spectral Shi cannot be used to identify kinetic constants (k off and k on ) yet. It also requires fluorescent labeling of one of the binding partners.