The Tm of AncFMO1 was assessed and determined using a Tycho NT.6 system (NanoTemper Technologies GmbH, Munich, Germany) in the absence and presence of 200-μM NADP+, respectively. Concentrations of AncFMO1 were determined using εFAD = 12.0 mM−1.cm−1 at 442 nm. Experiments were performed in triplicate, with each sample containing AncFMO1 (1.0 mg ml−1, determined using the calculated molecular weight of AncFMO1, 61 kDa), with or without NADP+ (200 μM), made to a final volume of 10 μl using the storage buffer. To ensure the Tm of AncFMO1 assessed using the Tycho NT.6 system was comparable to the ThermoFAD assay performed on the AncFMOs (23 (link)), a control experiment was performed using AncFMO2, which corroborated the previously observed Tm values (data not shown).
Tycho nt 6 system
Manufactured by NanoTemper
Sourced in Germany
The Tycho NT.6 system is a lab equipment product from NanoTemper. It is designed to perform high-throughput thermal stability and interaction analysis of biomolecules such as proteins, nucleic acids, and small molecules. The system utilizes microscale thermophoresis technology to measure target-ligand interactions and biomolecular thermal stability parameters.
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Lab products found in correlation
4 protocols using tycho nt 6 system
1
Determining AncFMO1 Thermostability with NADP+
2
Thermal Stability Evaluation by nanoDSF and Thermal Shift Assay
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Thermal stability was evaluated by nanoDSF using the Tycho NT.6 system (NanoTemper Technologies GmbH, München, Germany)67 (link). Also, Thermal stability at a constant 37 °C was measured by a thermal shift assay using a Mx3000p real-time PCR instrument (Agilent technologies, Santa Clara, California, USA) and SYPRO orange (Thermo Fisher Scientific)68 (link).
3
Thermal Stability of Exosomal Complexes
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Inflection temperature (Ti) was measured to compare the stability of exosomal subunits or complexes. Ti measurements were performed using a Tycho NT.6 system (Nanotemper). Proteins at a concentration of 1 mg/mL were prepared in PBS buffer, and loaded into capillaries (Nanotemper). While the samples were heated at a rate of 3 • C per minute between 35 • C and 95 • C, intrinsic fluorescence was detected at 330 nm and 350 nm. The ratio of fluorescence (350/330 nm) and the Ti were plotted by the Tycho NT.6 system.
4
Investigating Thermal Stability and Activity of proM Mutants
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To determine the effect of mutation on the thermal stability of proM, differential scanning fluorimetry was performed using the Tycho NT.6 system (NanoTemper, Munich, Germany). Briefly, proME225A, proME225A/C23A, proME225A/C23L, and mirolysin (8 µM) in 20 mM Tris, 5 mM CaCl2, 0.02% NaN3, pH 8.0 were transferred to glass capillaries, and unfolding profiles were obtained by measuring the fluorescence of intrinsic tryptophan and tyrosine residues detected at both 350 nm and 330 nm over a 30 °C/min temperature ramp in the range of 35–95 °C. The inflection temperature (Ti), representing the unfolding transition, was determined by NanoTemper software. Additionally, the activities of proMwt, proMC23A, proMC23L and mirolysin (10 nM) against FTC-casein (50 µg/ml) were determined.
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