Measurements were performed on the Prometheus NT.48 system (Nanotemper Technologies). Protein samples of 60 μM in phosphate buffer (20 mM Na2HPO4/NaH2PO4, pH 7.4, and 100 mM NaCl) and in the presence of different urea concentrations (from 0 to 8 M) were analyzed in Prometheus NT.48 Standard capillaries (Nanotemper Technologies).
Prometheus nt 48 system
Manufactured by NanoTemper
Sourced in Germany
The Prometheus NT.48 system is a laboratory equipment designed for biophysical analysis. It measures the thermal stability and binding properties of proteins and other biomolecules. The system uses nanoDSF (nano Differential Scanning Fluorimetry) technology to provide accurate and reliable data on the thermal unfolding of samples.
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9 protocols using prometheus nt 48 system
1
Unfolding Protein Thermal Stability
2
Thermal Stability Analysis of Proteins
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The nanoDSF method was performed on a Prometheus NT.48 system (Nano Temper Technologies, Munich, Germany). nanoDSF uses intrinsic fluorescence, which is a dye-free method to evaluate changes in aromatic residues (fluorophores) within proteins in response to the changes in their local environment. The shift and intensity change in fluorescence emission is monitored, with a change in the intrinsic fluorescence indicating that the protein has unfolded. Thermal stability of protein is characterized using the melting temperature (Tm), which indicates the point at which half the protein is unfolded. In the nanoDSF method, this is determined by using the ratio of fluorescence recorded at 330 nm and 350 nm; this ratio has shown to be more sensitive in detecting Tm as compared to the use of a single wavelength. Samples were filled in capillary tubes without any further preparation and excited at 285 nm with 20% power output. The thermal profiles were recorded from 20 to 95⁰ C with 2⁰ C/min scan rate.
3
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).
4
Thermal Stability Assay of drMHR1/2
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nDSF measurements were carried out on a Prometheus NT.48 system (Nanotemper). The 10 μM drMHR1/2 protein was mixed with varying amounts (600 nM to 2 mM) of ADPR or 2′‐deoxy ADPR in buffer M (see purification). Thermal unfolding was measured by following intrinsic tryptophan fluorescence during a thermal ramp (1°C/min). The PR.ThermControl software (Nanotemper) was used to determine melting temperatures. Binding parameter analysis was performed by simple Hill fit in the ORIGIN™ software.
5
Thermal Stability Profiling of Proteins
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NanoDSF measurements were carried out on a Prometheus NT.48 system (NanoTemper Technologies, Germany). Protein samples were diluted to 1 mg/ml in the buffer containing 25 mM Tris-HCl, pH 8.0, and 150 mM NaCl. Capillaries were then filled with samples individually and loaded into the instrument and heated from 20 °C to 85 °C at the heating rate of 1 °C/min. The intrinsic fluorescence emission at 330 nm and 350 nm was recorded. The melting temperature(Tm) was determined by the PR. ThermControl software (NanoTemper Technologies, Germany).
6
Nanoscale Thermal Unfolding Assay
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Nano-DSF thermal unfolding experiments were performed on a Prometheus NT.48 system (NanoTemper). Proteins were diluted to 8 μM sE in 1× PBS (pH 7.4), and 10 μl of each protein was loaded into a capillary. The protein samples were subjected to a heating regime of 1 °C/min from 20 to 95 °C. The intrinsic fluorescence signals at 330 and 350 nm were measured for each experiment. The Tm was calculated automatically based on the first derivative of fluorescence signal by the software PR.ThermControl (NanoTemper). The Tm values reported in this study were extrapolated from the first derivative of 350 nm fluorescence signal.
7
Thermal and Chemical Denaturation Analysis
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The experiments were conducted by using
the Prometheus NT.48 system (NanoTemper Technologies, Munich, Germany).
The fraction of folded and unfolded proteins was determined based
on the ratio of fluorescence changes at 350 and 330 nm. To determine
the Tm values, a sample of the protein
solution at 0.2 mg/mL (prepared by diluting the storage protein solution
with 200 mM MES buffer, pH 6.5) was subjected to a heating process
ranging from 25 °C to 85 °C at a heating rate of 1.0 °C/min.
For equilibrium denaturant titrations, 24 samples of the protein solution
at 0.2 mg/mL (prepared by diluting the storage protein solution with
200 mM MES buffer containing 0–4 M Gnd·HCl or 0–8
M urea, pH 6.5) were incubated at 4 °C for 18 h equilibrium,
and then fluorescence changes were measured at 25 °C.
the Prometheus NT.48 system (NanoTemper Technologies, Munich, Germany).
The fraction of folded and unfolded proteins was determined based
on the ratio of fluorescence changes at 350 and 330 nm. To determine
the Tm values, a sample of the protein
solution at 0.2 mg/mL (prepared by diluting the storage protein solution
with 200 mM MES buffer, pH 6.5) was subjected to a heating process
ranging from 25 °C to 85 °C at a heating rate of 1.0 °C/min.
For equilibrium denaturant titrations, 24 samples of the protein solution
at 0.2 mg/mL (prepared by diluting the storage protein solution with
200 mM MES buffer containing 0–4 M Gnd·HCl or 0–8
M urea, pH 6.5) were incubated at 4 °C for 18 h equilibrium,
and then fluorescence changes were measured at 25 °C.
8
Protein Thermal Stability Analysis
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Thermal stability of the proteins was assessed by NanoDSF using the Prometheus NT.48 system (NanoTemper Technologies, Germany) in triplicates. The purified proteins of interest were buffer exchanged into 20 mM sodium phosphate, pH 7.4 using a microcon-10 kDa centrifugal filter (Merck, USA) and loaded into standard capillaries (10 µL) of NanoDSF grade. These loaded samples were then subjected to programmed thermal ramping (1 °C/minute) from 20 °C to 90 °C. A dual UV detector was used to measure the fluorescence signal resulting from the thermal denaturation of the protein at 330 nm and 350 nm. The first derivative of the ratio between the two signals was used to determine the inflection point/s and the melting temperature (Tm) was calculated using the PR.ThermControl Software (NanoTemper Technologies).
9
Thermal Unfolding of Purified SHBG
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Five hundred nanomolar purified ursine and human SHBG was equilibrated with 0 or 50 μm DHT in triplicates. Thermal unfolding profiles from 20.0 to 95.0 °C and a temperature increase of 1.0 °C·min−1 were measured on a Prometheus NT.48 system (NanoTemper Technologies, München, Germany) using high‐sensitivity capillaries. The unfolding temperatures were determined as the peak maximum of the first derivative of the fluorescent ratio 350 nm/330 nm as a function of the temperature.
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