For thermal unfolding profiles, protein samples were diluted to 3.0–4.5mgmL−1 in buffer B, loaded on standard capillaries and subjected to a linear 20–95°C thermal gradient at 0.5°C min−1 rate using a Prometheus NT.48 Nano-DSF instrument (NanoTemper Technologies). The temperature-dependent shift in intrinsic fluorescence at emission wavelengths of 330 and 350 nm was measured, and inflection points of fluorescence transition corresponding to the Tm values were determined as the first derivative maxima of the fluorescence intensities ratio at the measured wavelengths (F330/F350). Protein-ligand interaction studies were performed as described by Vivoli and colleagues (Vivoli et al., 2014 (link)). Myricetin (≥96%, Sigma-Aldrich) and TBBT (≥98%, Sigma-Aldrich) were tested at fixed concentration in excess (100 μM Cf) or equal to the IC50 value obtained in the in vitro split-GFP FC assay, and against a fixed amount of protein. After incubation of 60minat RT, sample quadruplicates or triplicates were tested and Tm values were averaged from at least three independent measurements, then processed using the PR.ThermControl software (NanoTemper Technologies).
Thermcontrol software
Manufactured by NanoTemper
Sourced in Germany
ThermControl is a software suite developed by NanoTemper to control and analyze data from their thermal analysis instrumentation. It allows users to configure instrument parameters, monitor experiments in real-time, and perform data analysis. The software provides a user-friendly interface for managing thermal experiments and processing the resulting data.
Automatically generated - may contain errors
Lab products found in correlation
Prometheus nt 48, by NanoTemper (6 mentions)
Standard grade capillaries, by NanoTemper (2 mentions)
Nano dsf prometheus nt 48, by NanoTemper (2 mentions)
Prometheus nt 48 nanodsf instrument, by NanoTemper (1 mentions)
Myricetin, by Merck Group (1 mentions)
Timecontrol software, by NanoTemper (1 mentions)
Nanodsf grade standard capillaries, by NanoTemper (1 mentions)
Prometheus nt 48 system, by NanoTemper (1 mentions)
Prometheus nt 48 device, by NanoTemper (1 mentions)
15 protocols using thermcontrol software
1
Thermal Unfolding and Ligand Binding Assays
2
Thermal Unfolding of FUS-SNAP
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Thermal unfolding of FUS-SNAP was performed using nanoDSF with a Prometheus NT.48 (NanoTemper Technologies, München, Germany) instrument. Protein samples were prepared in buffers and centrifuged shortly (5 min, 10,000 RCF) to remove protein aggregates. Samples were loaded into high-sensitivity glass capillaries (Cat#PR-C006, NanoTemper Technologies, München, Germany) and exposed at a linear thermal ramp from 20 °C to 95 °C by thermal ramping rate of 1 °C/min. Intrinsic protein fluorescence emission was collected at 330 and 350 nm with a dual-UV detector over a temperature gradient. The fluorescence intensity ratio (350/330) was plotted against the temperature, and the inflection point of the transition was derived from the maximum of the first derivative for each measurement using Therm-Control Software (NanoTemper Technologies, München, Germany). All experiments were carried out in triplicate; mean and standard deviation were calculated for all three measurements.
3
Thermostability Analysis of Biomolecules
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Thermostability tests were performed using a nano-DSF Prometheus NT.48 instrument and standard grade capillaries (Nano-Temper Technologies). In each instance, the samples at ∼0.2 mg/ml were subjected to a temperature variance of 20°C to 95°C, using a thermal ramp of 1°C per minute. Values reported correspond to the inflection point calculated within the PR.ThermControl software (Nano-Temper Technologies).
4
Thermal Stability Profiling of Proteins via NanoDSF
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NanoDSF was performed using Prometheus NT.48 equipped with backreflection mode (NanoTemper Technologies, München, Germany). Samples were loaded in nanoDSF grade standard capillaries (NanoTemper Technologies GmbH, München, Germany) and exposed at thermal stress from 20 °C to 95 °C by thermal ramping rate of 1 °C/min. Fluorescence emission from tryptophan after UV excitation at 280 nm was collected at 330 nm and 350 nm with dual-UV detector. Protein aggregation was assessed simultaneously employing backreflection optics, which detects protein aggregation by measuring the attenuation of backreflected light intensity passing through the sample. Thermal stability parameters, including Tonset, Tm, and Tagg, were calculated by PR.ThermControl software (NanoTemper Technologies, München, Germany). For isothermal stability, the time interval data from thermal stress at constant temperature (50, 54, 57, or 60 °C) were collected by PR.TimeControl software (NanoTemper Technologies, München, Germany).
5
Rapid Protein Stability Assessment with nanoDSF
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NanoDSF is a microscale label-free method for rapid and easy detection of protein stability using the intrinsic aromatic amino acids tryptophane and tyrosine to determine protein folding and stability [38 (link),39 (link)]. We used the nanoDSF technique to analyze both the stability of purified SdiA and putative binding of several compounds such as AHLs (10 nM C4-AHL, 10 nM C8-AHL, and 10 nM C12-AHL), 3.3% (v/v) PRE and their respective HPLC-separated fractions to SdiA via protein stability, assuming that ligand binding affects melting temperature of the protein. For that purpose, the protein sample was adjusted to a concentration of 0.3 mg/mL and loaded into capillaries, which were placed into a Prometheus NT.48 (NanoTemper Technologies GmbH, München, Germany) device. The measurement was performed in a temperature range between 20 °C and 90 °C with a temperature slope of 1.5 °C/min. The resulting data were analyzed using the PR.ThermControl software (NanoTemper).
6
Thermal Stability Analysis of Proteins
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All nanoDSF experiments were performed on a Prometheus NT.48 system (NanoTemper Technologies, Germany) in triplicate. First, protein samples were diluted to a 1 mg/mL concentration in buffer containing 25 mM Tris-HCl, pH 8.0, and 150 mM NaCl. Next, capillaries filled with samples were placed on the loading tray and heated from 20°C to 85°C at a heating rate of 1°C/min. The fluorescence at 330 and 350 nm and the light scattering signals were recorded. The melting temperature (Tm) and the aggregation temperature (Tagg) were determined by the PR. ThermControl software (NanoTemper Technologies, Germany).
7
Thermal Stability Analysis of scFvs
8
Thermal Stability Analysis of Anti-VEGF Biologics
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To determine the thermal stability of the anti-VEGF biologics, nano-DSF analysis was performed on a Prometheus NT.48 (NanoTemper Technologies GmbH). Undiluted samples were drawn into capillaries and run in triplicates. The instrument was set to gradually increase the temperature from 20 °C to 95 °C. As the temperature increased, the ratio between 330 nm and 350 nm wavelengths was plotted against temperature. The melting temperature (Tm) for which half of the proteins were unfolded was determined by deducing the first derivative in the PR.ThermControl software (NanoTemper Technologies GmbH).
9
Thermal Unfolding Analysis of Human UCP1
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Thermal unfolding analysis was performed using dye-free differential scanning fluorimetry (nanoDSF) (29 ). Human UCP1 has two tryptophan residues: W174 and W281. Approximately 5 μg of protein was added into a final volume of 10 μl of buffer [100 mM MES/Hepes (pH range from 6.0 to 8.0), 50 mM NaCl, 0.02% decyl maltose neopentyl glycol, and tetraoleoyl cardiolipin (0.02 mg/ml)] and, when required, 1 mM compound. The samples were loaded into nanoDSF-grade standard glass capillaries. The temperature was increased by 4°C every minute from 25° to 95°C, the intrinsic fluorescence was measured in a Prometheus NT.48 nanoDSF device, and the apparent Tm was calculated with the PR.ThermControl software (NanoTemper Technologies).
10
Thermal Stability Analysis of IgA2
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NanoDSF was performed using Prometheus equipped with back reflection mode (NanoTemper Technologies, München, Germany). SEC-purified monomeric IgA2 (m2) were loaded in nanoDSF grade standard capillaries and exposed at temperatures from 15°C to 90°C using a thermal ramping rate of 1°C/min. Fluorescence emission from tryptophan after UV excitation at 280 nm was collected at 330 nm and 350 nm with a dual-UV detector. Thermal stability parameters were calculated using the ThermControl software (NanoTemper Technologies, München, Germany).
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