MST Monolith NT.115

Description

Microscale Thermophoresis Monolith NT.115 (NanoTemper) measures the motion of molecules along microscopic temperature gradients and detects changes in their hydration shell, charge or size. By combining the precision of fluorescence detection with the flexibility and sensitivity of thermophoresis, MST provides a flexible, robust and fast way to measure molecular interactions.

The equipment is accessible to both internal and external users upon prior request. Individuals interested in utilizing the equipment should contact the device administrator. Only trained operators can schedule the use of the equipment, or alternatively, the measurement can be conducted by the experienced staff of the Department of Molecular Biology and Nanobiotechnology.

Specifications

Measurements	Affinity (K_D), enthalpy (âˆ†H), stoichiometry, enzyme kinetics
Sample/experiment	16
Fluoroscent chanells	2 (red, green)
Range	nM–mM
Labelling	yes
Fluorescent molecule concentration	10^-9–10^-3
Complex media measurements	yes
Sample volume	4 μl
Sample mass	10 Da–10⁴ kDa
Experiment and analysis time	few minutes
Immobilization	not needed
Temperature range	22–45 °C
Maintenance	not needed

Sample requirements

Purity/quality of the sample needed	Pure samples are required and concentrations determined as accurately as possible (spectrophotometrically).
Amount needed for a typical measurement	Target molecule can be fluorescently labelled on-site. Concentration of unlabelled protein to use with a labelling kit: 100 μl of 200 nM (His-tag dye), 100 μl of 2–20 µM (amino-reactive and maleimide-reactive dyes). Approx. 200 μl of the labelled target is needed per one titration together with 20 μl of the unlabelled ligand molecule with a concentration 100-fold above the expected K_D (and not below 100 nM).
Buffer considerations	MST optimized buffer: 50 mM Tris pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 0.05 % Tween-20 or 0.1 % Pluronic F 127. For the His-tag labelling it is advised to use PBS with 0.05 % Tween-20. For the labelling with an amino-reactive dye, purified protein should be in a buffer that does not contain primary amines (e.g., ammonium ions, Tris, glycine, ethanolamine, glutathione) or imidazole. DTT and 2-mercaptoethanol interfere with the labelling reaction and therefore need to be avoided.
Other requirements	Guest researchers are kindly asked to fill in the Sample request form regarding sample quality, experimental conditions and plausible previous results. Buffers and consumables for experiments can be provided by the National Institute of Chemistry by prior arrangement.

Contact

Kristina Elersic Filipic

kristina.elersic.filipic@ki.si

Kemijski inštitut

Hajdrihova ulica 19, 1000 Ljubljana

Documents

Specifications
Protein-Small Molecule Interaction Analysis
Protein-Protein Interaction Analysis in Different Buffer Systems
Protein-DNA Interaction Analysis