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.

Specifications

Measurements Affinity (KD), enthalpy (∆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 KD (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

Katja Pirc

katja.pirc@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