Each sample was used to fill two standard grade nanoDSF capillaries (Nanotemper, Germany) and loaded into a Prometheus NT.48 device (Nanotemper) controlled by the PR.ThermControl v.2.1.2 software. Excitation power was pre-adjusted to get fluorescence readings above 2000 RFU for F330 and F350, and samples were heated from 15 to 95 °C with a slope of 1 °C/min. An XLSX file containing the processed nanoDSF data was exported from PR.ThermControl and used for further analysis to extract the melting (Tm) and onset denaturation (Tonset) temperatures (Kotov et al., 2019 (link)). Normalization of curves to calculate the fraction of unfolded protein was performed using the MoltenProt software (Kotov et al., 2021 (link)).
Nanodsf grade standard capillaries
Manufactured by NanoTemper
Sourced in Germany
NanoDSF grade standard capillaries are specialized consumable items designed for use with NanoDSF instruments. They are engineered to provide accurate and reliable sample containment for thermal analysis applications.
Automatically generated - may contain errors
Lab products found in correlation
Prometheus nt 48 nanodsf device, by NanoTemper (5 mentions)
Prometheus nt 48, by NanoTemper (5 mentions)
Prometheus nt 48 device, by NanoTemper (3 mentions)
Pr thermcontrol, by NanoTemper (2 mentions)
Prometheus nt 48 instrument, by NanoTemper (1 mentions)
Thermcontrol software, by NanoTemper (1 mentions)
Timecontrol software, by NanoTemper (1 mentions)
Prometheus nt plex nanodsf instrument, by NanoTemper (1 mentions)
Pr thermcontrol software, by NanoTemper (1 mentions)
16 protocols using nanodsf grade standard capillaries
1
Thermal Unfolding Analysis of Proteins
2
Thermal Stability Analysis of SARS-CoV-2 Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The thermal stability of RBD, ACE2, and spike was monitored by nanoDSF (NanoTemper Technologies) at a concentration of 0.25 mg/ml in PBS (10 mM phosphate, 2.7 mM KCl and 137 M NaCl, pH 7.4) buffer. To monitor binding-induced thermal shift changes of the RBD with different sybodies, RBD at 0.25 mg/ml was incubated with 0.15 mg/ml of different sybodies (Sb23, Sb76, Sb95, control Sb) and incubated for at least 10 min at RT prior analysis. Standard grade nanoDSF capillaries (NanoTemper Technologies) were loaded into a Prometheus NT.48 device (NanoTemper Technologies) controlled by PR. ThermControl (version 2.1.2). Excitation power was adjusted to 30% and samples were heated from 20 °C to 90 °C with a slope of 1 °C/min. All samples were run in triplicates and error bars represent standard deviations. The stability of the different sybodies was analyzed in independent experiments and they all display transition temperatures in the range of 70–95 °C.
3
Thermal Stability Characterization by nanoDSF
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The assay was carried out in 25 mM Tris-HCl (pH
7.5, 0.5 M NaCl) buffer with the protein and ligands concentration
preserved constant at 2.5 and 25 μM, respectively. The samples
were loaded into nanoDSF grade Standard Capillaries (NanoTemper Technologies)
and analyzed using the Prometheus NT.48 nanoDSF device (NanoTemper
Technologies). Thermal unfolding of the protein was monitored using
a linear thermal ramp (1 °C·min–1; 20–80
°C) with an excitation power of 30%. All numerical models were
globally fitted to the experimental data, assuming a two-state cooperative
transition at Tm, using the Marquardt
algorithm52 (link) implemented in the Origin 2019
package (OriginLab, Northampton, MA;www.originlab.com ) according
to the following equations. where F(T) is the observed fluorescence
signal, Ffold(T) and Funf(T) are the low- and high-temperature
linear asymptotes of F(T); ΔG(T) is the free energy of unfolding at
a given temperature; Tm is the middle-point
transition temperature;
ΔHTm and ΔSTm are the heat and entropy
of the unfolding at Tm (ΔG(Tm) = 0); and ΔCp is the heat capacity change
upon the protein unfolding.53 (link)
7.5, 0.5 M NaCl) buffer with the protein and ligands concentration
preserved constant at 2.5 and 25 μM, respectively. The samples
were loaded into nanoDSF grade Standard Capillaries (NanoTemper Technologies)
and analyzed using the Prometheus NT.48 nanoDSF device (NanoTemper
Technologies). Thermal unfolding of the protein was monitored using
a linear thermal ramp (1 °C·min–1; 20–80
°C) with an excitation power of 30%. All numerical models were
globally fitted to the experimental data, assuming a two-state cooperative
transition at Tm, using the Marquardt
algorithm52 (link) implemented in the Origin 2019
package (OriginLab, Northampton, MA;
to the following equations. where F(T) is the observed fluorescence
signal, Ffold(T) and Funf(T) are the low- and high-temperature
linear asymptotes of F(T); ΔG(T) is the free energy of unfolding at
a given temperature; Tm is the middle-point
transition temperature;
ΔHTm and ΔSTm are the heat and entropy
of the unfolding at Tm (ΔG(Tm) = 0); and ΔCp is the heat capacity change
upon the protein unfolding.53 (link)
4
Thermal Stability Analysis of MEK1 Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To compare stability of NT647-MEK1 and unmodified MEK1 protein, thermal unfolding profiles of the proteins were recorded using the Prometheus NT.48 instrument (NanoTemper Technologies). For this, 30 µL of a 2 µM solution of each protein in assay buffer was prepared, and 3 × 10 µL was loaded into nanoDSF grade standard capillaries (NanoTemper Technologies) for triplicate measurements. Thermal unfolding of triplicates was analyzed in a thermal ramp from 25 to 80 °C with a heating rate of 1 °C/min. Unfolding transition temperatures (Tm) were automatically determined by the software and represented as mean ± SD.
5
Determining Cas9 Protein Melting Point
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified Cas9 proteins were diluted in 300 mM NaCl, 20 mM Tris, pH 7.5 to 5–10 µM at room temperature. In total, 10 µL of the diluted protein was loaded into NanoDSF Grade Standard Capillaries (NanoTemper), and melting temperatures were determined using a Prometheus NT4.8 NanoDSF instrument according to the manufacturer’s instruction. The temperature was increased from 20 °C to 80 °C at a rate of 1 °C/min. Inflection points of melting curves are reported as the Tm.
6
Thermal Stability Analysis of Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prometheus NT.48 instrument (NanoTemper Technologies) performed DSF to determine Tm and Tonset of loaded protein samples. Purified protein samples in 20 mM sodium phosphate, pH 7.4, 300 mM NaCl, 500 mM imidazole were loaded in nanoDSF grade standard capillaries (NanoTemper Technologies) and laser power optimized for signal. Thermal stress from 20 to 95°C was performed on the sample shifting at a rate of 1°C/min. Fluorescence emission from the single Ist1 tryptophan (W134) after excitation with UV at 280 nm was collected at 330–350 nm. Aggregation of protein was assessed concurrently with back reflection optics. Thermal stability parameters Tm and Tonset were unbiasedly assessed and calculated by PR. ThermControl software (V.2.1.3).
7
Thermal Stability Profiling of Proteins via NanoDSF
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
8
Thermal Stability Analysis of Protein-Ligand Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The assay was carried out in 25 mM Bis–Tris Propane, 0.5 M NaCl buffer with constant protein and ligand concentrations of 2.5 μM and 25 μM, respectively. The samples were loaded into nanoDSF Grade Standard Capillaries (NanoTemper Technologies) and analyzed using the Prometheus NT.48 nanoDSF device (NanoTemper Technologies). All experiments were performed and analyzed as described previously65 (link).
9
Thermal Unfolding of Cyani HAP2 Ectodomain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cyani HAP2 ectodomain purified from Expi293F cells (20 mg/mL in 1 μL 20 mM Tris-HCl, pH 7.5, 500 mM NaCl was diluted with 19 μL of buffers containing 500 mM NaCl and the following: 50 mM HCI-KCI (pH 1.5 and pH 2.0), citric acid-trisodium citrate (pH 3.5, pH 4.3, and pH 5.0), or Tris-HCl (pH 7.5). Duplicate samples in nanoDSF Grade Standard Capillaries (NanoTemper Technologies) were subjected to thermal unfolding in a Prometheus NT.Plex nanoDSF instrument (NanoTemper Technologies) with a linear thermal ramp of 2 °C/min from 20 °C to 95 °C with excitation at 275 nm at a power of 30%. The fluorescence intensity ratio (FIR) of tryptophan emission at 350 nm/330 nm was determined.
10
Thermal Stability Analysis of Ca2+-Loaded Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Thermal stability assays were performed by nanodifferential scanning fluorimetry (nanoDSF) using a Prometheus NT.48 instrument (NanoTemper Technologies, Germany). The Ca2+-loaded protein samples (0.2 mg/ml) were diluted in 5 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 1.5 mM CaCl2 and loaded into nanoDSF grade standard capillaries (NanoTemper Technologies, Germany). The measurements were conducted from 20 to 95 °C (with a temperature ramp of 2 °C/min) under constant monitoring of tryptophan fluorescence at 350 and 330 nm. The melting temperature (Tm) values, corresponding to the inflection points of the unfolding curve, were determined by using a PR.ThermControl (NanoTemper Technologies, Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!