MST Monolith NT.115
Opis
Aparatura Monolith NT.115 (NanoTemper) meri spremembe v gibanju molekul v temperaturnem gradientu, ki nastanejo po osvetlitvi vzorcev z laserskim žarkom. En partner proučevane interakcije mora biti označen s fluorescenčno molekulo. Mikrotermoforeza je občutljiva na spremembe velikosti, naboja ali konformacije, ki se lahko zgodijo ob vezavi molekul. Lahko se meri vezavo tudi manjših molekul ali celo ionov.
Lastnosti
| Meritve | afiniteta (KD), entalpija (∆H), stehiometrija, encimska kinetika |
| Število vzorcev/eksperiment | 16 |
| Fluoroscenčni kanali | 2 (rdeč, zelen) |
| Območje meritev | nM-mM |
| Potrebno označevanje | da |
| Koncentracija fluorescenčne molekule | 10-9-10-3 |
| Meritve v kompleksnem mediju | da |
| Volumen vzorca | 4 μl |
| Velikost vzorcev | 10 Da–104 kDa |
| Čas za eksperiment in analizo | nekaj minut |
| Imobilizacija | ni potrebna |
| Temperaturno območje | 22–45 °C |
| Vzdrževanje | ni potrebno |
Priprava vzorcev
| 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. |