Spectral Shift leverages the power of fluorescence to detect minute variations, known as spectral shifts, in the wavelengths of light emitted by a fluorophore that is attached to a biomolecule (the “target”). By monitoring these spectral shifts, scientists can get valuable insights into the strength and nature of the interaction between the target molecule and its ligand (binding partner).
The brilliance of Spectral Shift lies in its capability to effectively measure interactions that often prove troublesome for other techniques. This is particularly true for interactions involving multimeric complexes, intrinsically disordered proteins (IDPs), covalent ligands, and molecules unsuitable for biosensor immobilization.
The choice of dye, properties of the target and ligand molecules, and successful labeling of the target significantly impact the sensitivity and accuracy of Spectral Shift measurements. 2bind's team of experts has extensive experience in selecting the right dyes for diverse biomolecules, along with performing efficient and reliable labeling procedures. We ensure the best possible detection limits for your specific interaction, eliminating the need for in-house labeling efforts.
Equilibrium dissociation constant. Can be obtained by kinetic or classical equilibrium binding analysis. Provides information about the strength but not the dynamics of an interaction.
Binding enthalpy. KD values at different temperatures can be used to obtain the binding enthalpy of an interaction via van't-Hoff-plots.
Fast screening for qualitative (Yes/No) binding.
Determination of competition of several ligands for a selected binding site on a target.
Determination of binding site blocking by a ligand and prevention of binding of a natural interaction partner.
Determination of equilibrium binding stoichiometry of target-ligand complex formation.
Spectral shift analysis is a powerful biophysical technique used to precisely measure molecular interaction strength (affinity). How does it work? When a ligand molecule binds, it triggers subtle variations in a labeled molecule’s fluorescence spectrum – think blue shifts, red shifts, or emission intensity changes. Ligands that bind close to the fluorophore can directly influence the fluorophore’s chemical environment. Ligands that bind in a distant position from the fluorophore can lead to ligand-induced conformational changes that affect the chemical environment of the fluorophore. For measuring Spectral Shift, a fluorescent dye is attached to the target molecule, for example by covalent coupling via NHS or Maleimide chemistry, or via affinity-based tags, like a His-tag dye.
Spectral Shift measurements are done either in a glass capillary setup (Monolith-series instruments) or in a plate-based format (Dianthus). In both setups, the fluorescence of the dye that is attached to the target molecule is excited by a laser and emission is monitored at two wavelengths (650 and 670 nm). The capillaries or wells each contain a mix of fluorescent binding partner (usually called the “target”) and non-fluorescent binding partner, which is titrated in a dilution series (usually called the “ligand”).
The sample fluorescence in each capillary or well is measured for a short time-span to allow for a statistically sound determination of the spectral shifts. Then, the ratio between the fluorescence signal in the 670 nm channel and the fluorescence signal in the 650 nm channel is calculated and plotted against the ligand concentration. The resulting dose-response curve is then fitted to the law of mass equation to yield the affinity constant (KD) of the molecular interaction.
Since detection of the spectral shifts happens at 650 and 670 nm, “red” wavelength spectrum fluorescent dyes are required for Spectral Shift. Commonly, specialized Spectral Shift-dyes are used, which maximize the obtainable response. However, also standard dyes like Cy5 work well with Spectral Shift, for example when used for DNA- or RNA-labeling. Fluorescent labeling of your target molecule is performed by 2bind and included in most projects. Alternatively, many oligonucleotide- or peptide-based targets can also be synthesized with appropriate labels already attached.
No, Spectral Shift requires the presence of a highly sensitive fluorescent dye to pick-up minute changes in the fluorescence properties upon target-ligand interaction. This would not be possible by harnessing for example a protein-intrinsic Tryptophan fluorescence.
No, for protein targets, 2bind performs all required steps for fluorescence labeling of the target. Peptides, DNA, or RNA targets are often synthesized directly with a selected fluorescent dye attached.
The most common labeling methods are covalent NHS- or Maleimide-chemistry reactions. These reactions attach fluorescent dyes directly to lysine or cysteine residues of the target protein, respectively. This is a routine part of the assay process at 2bind. The advantage of these covalent dye labels is the option to use very low (single-digit nM) concentrations of the labeled target protein and therefore determine even sub-nM KD values accurately. An alternative to covalent labelings are site-specific labelings at a His-tag, SNAP-tag, or biotinylated Avi-tag of the target protein.
Spectral Shift is perfect for measuring bi-molecular interactions between many kinds of biomolecules: proteins, peptides, DNA, RNA, aptamers, small molecules, fragments, lipids, carbohydrates, ions, or even large artificial or virus(like) particles. Spectral Shift especially excels for challenging target types like multimeric protein complexes or intrinsically disordered proteins (IDPs). Also, challenging ligand molecules like covalent binders, PROTACs, or molecular glues can reliably be analyzed with Spectral Shift.
Spectral Shift mainly determines the equilibrium, steady-state binding affinity of two molecules (KD). In addition, Spectral Shift can also be used to assess parameters such as stoichiometry, aggregation, precipitation, enthalpy (van’t Hoff plot), slow enzyme kinetics, and oligomerization.
2bind offers the following assay types with Spectral Shift: Hight-throughput interaction screenings, steady-state binding affinity assays, steady-state binding affinity assays in biological liquids, complex formation-inhibition assays, competition assays, binding assays with multiple binding partners (e.g. ternary complex formation).
Spectral Shift offers many advantages compared to other methods for determining the affinity of a molecular interaction:
Spectral Shift determines the equilibrium, steady-state binding affinity of two molecules (KD). Therefore, samples are analyzed after the binding reaction reaches chemical equilibrium. Measurement of kinetics is only possible if the equilibrium is not reached quickly. The thermodynamics of an interaction can be investigated using the van’t Hoff method. However, for in-depth kinetic analysis of molecular interactions other technologies like GCI or BLI are excellent. For thermodynamics, an ITC is preferred.
Absolutely! Due to the very fast measurement nature of Spectral Shift, time-series can be recorded that show the continuous association and finally covalent binding of such ligands.
Yes, due to its inherently robust physical principle and technical readout (specific fluorescent dye on the target molecule and calculation of a ratio signal between two wavelengths), Spectral Shift can be measured even in 100% complex bioliquids like serum, plasma, cell lysate, urine, saliva, sea water, etc.
