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). Thermodynamic parameters such as ΔH and ΔS provide valuable insights into complex formation and possible conformational changes of a binding partner after the binding event.

The Bioanalytic Core Facility offers the usage of the PEAQ-ITC microcalorimeter from Malvern. For more details about our PEAQ-ITC instrument, please visit the MalvernPanalytical

Mobile particles exhibit different responses to the force of a temperature gradient. This ohenomenon is called thermophoresis. The Bioanalytics core facility is equipped with a Monolith NT.115 device (NanoTemper) Microscale-Thermophoresis device that is based on detection of fluorescent proteins or dyes.

Therefore, affinities of biomolecular interactions of dye-labelled proteins or DNA e.g. with any non-labelled compound and can easily be measured in solution in any buffer or complex bioliquid, using only a tiny amount of sample. We offer training at the instrument and help interpreting the data. Usage is at cost, so users only have to pay consumables (glass capillaries) that have to be bought by themselves.

Please use the Nanotemper online shop to purchase consumables

Biacore systems define the characteristics of proteins in terms of their specificity of interaction with other molecules, the rates at which they interact (binding and dissociation), and their affinity (how tightly they bind to another molecule). Binding can be monitored in real-time in a labelfree system. We use the latest technology for measurements on the Biacore T200 system. We support your project in technical and scientific questions.

BInteractions between biomolecules play a central role in all cellular processes. These can be, for example, interactions between protein-protein, DNA-protein, lipid-protein, or protein-low molecular weight ligand. In order to understand the function of proteins, nucleic acids, lipids or other molecules in a biological system, it is essential to know their interaction partners and to characterize their interactions.

The characterization of biomolecular interactions requires knowledge of the affinity, the number of binding sites and the thermodynamic properties. Using a simple ITC experiment, it is possible to determine parameters such as binding affinity (KD), stoichiometry, enthalpy (ΔH) and entropy (ΔS).

Knowledge of thermodymic parameters such as ΔH and ΔS provides further information about complex formation, but also about possible conformational changes of a binding partner after the binding event. This is possible with the PEAQ-ITC microcalorimeter from Malvern; the device is located in the Bioanalytics service unit.

The characterization of isolated biomolecules is an essential part of molecular biological research studies. For example, in order to understand how proteins function, it is necessary to know their exact biophysical properties.
By combining MALS (“Multiple Angle Light Scattering”) and DLS (“Dynamic Light Scattering”), the absolute molar mass of biomolecules as well as their radii can be determined precisely and without calibration, so that clear statements can be made about the oligomerization state and the conformation of macromolecules such as proteins, other natural biopolymers or nanoparticles.

However, this knowledge allows conclusions to be drawn about the aggregation, stability and homogeneity of biomolecules and is therefore extremely important for the optimal preparation of samples for subsequent studies such as biomolecular interaction studies or crystallization. The Bioanalytics Service Unit is equipped with a DLS detector (Wyatt DynaPro Nanostar) and a MALS detector (Wyatt miniDAWN TREOS). The combination of both techniques offers an extremely sensitive measurement method to determine the above-mentioned biophysical properties of e.g. proteins.

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 and help interpreting the data. Usage is at cost, so users only have to pay consumables (gas/standards, payed by usuage per hour) and a maintainance fee (fixed fee payed per measurement).

Fluorescence spectroscopy is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light of a lower energy, typically, but not necessarily, visible light.

Conformational changes of a protein (e.g. by binding a ligand) can be investigated changes in the fluorescence spectrum. The fluorescence of a folded protein is a mixture of the fluorescence from individual aromatic residues. Most of the intrinsic fluorescence emissions of a folded protein are due to excitation of tryptophan residues, with some emissions due to tyrosine and phenylalanine; but disulfide bonds also have appreciable absorption in this wavelength range.

Typically, tryptophan has a wavelength of maximum absorption of 280 nm and an emission peak that is solvatochromic, ranging from ca. 300 to 350 nm depending in the polarity of the local environment. Hence, protein fluorescence may be used as a diagnostic of the conformational state of a protein.

Furthermore, tryptophan fluorescence is strongly influenced by the proximity of other residues (i.e., nearby protonated groups such as Asp or Glu can cause quenching of Trp fluorescence). Also, energy transfer between tryptophan and the other fluorescent amino acids is possible, which would affect the analysis, especially in cases where the Förster acidic approach is taken. In addition, tryptophan is a relatively rare amino acid; many proteins contain only one or a few tryptophan residues.

Therefore, tryptophan fluorescence can be a very sensitive measurement of the conformational state of individual tryptophan residues. The advantage compared to extrinsic probes is that the protein itself is not changed. The use of intrinsic fluorescence for the study of protein conformation is in practice limited to cases with few (or perhaps only one) tryptophan residues, since each experiences a different local environment, which gives rise to different emission spectra.

We offer training at the instrument and help interpreting the data. Usage is at cost, so users only have to pay a maintainance fee (fixed fee payed per measurement).