MST Monolith NT.115
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.
|Measurements||Affinity (KD), enthalpy (∆H), stoichiometry, enzyme kinetics
|Fluoroscent chanells||2 (red, green)|
|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|
|Temperature range||22–45 °C|
|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).|
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.
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.