Bioanalytic

Interactions between biomolecules play a crucial role in various cellular processes. These interactions can involve protein-protein, DNA-protein, lipid-protein, or protein-low molecular weight ligand interactions. To understand the functioning of proteins, nucleic acids, lipids, and other molecules in a biological system, it is essential to identify their interaction partners and characterize their interactions. The characterization of biomolecular interactions requires knowledge of affinity, the number of binding sites, and thermodynamic properties.

By performing a simple ITC experiment, it is possible to determine parameters such as binding affinity (KD), stoichiometry, enthalpy (ΔH), and entropy (ΔS). hese thermodynamic parameters provide valuable insights into complex formation and conformational changes that may occur upon binding.

The Bioanalytic Core Facility provides access to the PEAQ-ITC microcalorimeter (Malvern).

Mobile particles exhibit different responses to the force of a temperature gradient. This phenomenon is called thermophoresis. The Bioanalytic Core Facility is equipped with a Monolith NT.115 (NanoTemper) for microscale thermophoresis measurements, which detect the movement of fluorescently labeled molecules in response to temperature changes.

This technique allows the determination of affinities between dye-labeled molecules, such as proteins or DNA, and unlabeled binding partners in virtually any buffer or complex liquids, using only minimal sample volumes. We provide training on the instrument and support with data interpretation.

Users are required to purchase consumables, such as capillaries or labeling kits, directly from the NanoTemper online shop.

The Prometheus NT.48 from NanoTemper enables label-free analysis of protein stability using nanoDSF (nanodifferential scanning fluorimetry). This technique monitors changes in the intrinsic tryptophan fluorescence of proteins during controlled heating, providing precise information on thermal stability and unfolding transitions. Only small sample volumes are required, and no special cuvettes or dyes are needed, making nanoDSF a fast and efficient tool for assessing protein quality.

Users must purchase the appropriate capillaries themselves via the NanoTemper online shop.

Biomolecular interactions are often defined by their kinetics and affinities, which provide critical insights into molecular regulation and mechanisms. These interactions can involve proteins, nucleic acids, lipids, or small molecules, and require not only the measurement of binding strength but also of association and dissociation rates.

SPR technology enables real-time, label-free monitoring of such interactions. Using the Biacore T200 (Cytiva), it is possible to determine parameters such as association and dissociation rates (kon and koff), equilibrium binding affinity (KD), and binding specificity. This is particularly valuable for studying weak or transient interactions that are challenging to capture with other methods.

The Bioanalytic Core Facility provides access to the Biacore T200 and offers both collaborative research opportunities and full-service support. Please contact us to discuss how we can assist with your interaction studies.

Understanding the composition, oligomeric state, and binding behaviour of biomolecules is essential for elucidating their function in biological systems. Mass photometry is a powerful, label-free technique that enables accurate mass measurements of individual molecules in solution, under native conditions and without the need for labelling.

This method allows for quantitative single-molecule analysis of proteins, nucleic acids, and their complexes. It is also well-suited for assessing sample heterogeneity, determining the stoichiometry of molecular assemblies, and studying complex formation and dissociation.

The Bioanalytics core facility offers the TwoMP (Refeyn) for mass photometry to enable precise mass determination of single molecules by detecting their light scattering signals. Within minutes, and using only minimal sample volumes and low concentrations, the instrument can measure the mass distribution of biomolecules and quantify the relative abundance of different species.

These features make mass photometry an ideal tool for evaluating sample purity, oligomeric states, and molecular interactions with high sensitivity and minimal sample preparation.

Atomic Absorption Spectroscopy (AAS) uses the absorption of light to measure the concentration of gas-phase atoms. Since samples are usually liquids or solids, the analyte atoms or ions must be vaporized in a flame or graphite furnace. The atoms absorb ultraviolet or visible light and make transitions to higher electronic energy levels. The analyte concentration is determined from the amount of absorption.

Applying the Beer-Lambert law directly in AA spectroscopy is difficult due to variations in the atomization efficiency from the sample matrix, and nonuniformity of concentration and path length of analyte atoms (in graphite furnace AA). Concentration measurements are usually determined from a working curve after calibrating the instrument with standards of known concentration.

The light source is usually a hollow-cathode lamp of the element that is being measured.

To date we have the following lamps:

· Na/Li/K

· Multielement lamp: Co, Cr, Cu, Fe, Mn, Ni

· Cs

We offer training at the instrument Varian240 and help interpreting the data. Usage is cost-based, with fees covering consumables (gases, standards, billed by usage) and a fixed maintenance charge per measurement.

The FluoroMax3 spectrometer allows sensitive fluorescence measurements to investigate protein conformational changes, such as those induced by ligand binding. Since most intrinsic fluorescence arises from tryptophan residues, this technique provides a label-free way to monitor structural changes.

There are, however, important limitations. Interpretation can be challenging when proteins contain multiple tryptophan residues in different environments, and signals are easily influenced by quenching or energy transfer effects. In addition, measurements require quartz cuvettes and relatively large sample volumes, which may not always be practical. For these reasons, intrinsic fluorescence is most informative when applied to proteins with few tryptophan residues.

At the Bioanalytic Core Facility, we provide training on the FluoroMax3 and offer support with data interpretation.