Protein thermal shift software v1

Manufactured by Thermo Fisher Scientific

Sourced in United States

Protein Thermal Shift software v1.4 is a data analysis tool designed for the thermal shift assay. It provides functionality to process and analyze thermal shift data acquired from various experimental setups.

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59 protocols using protein thermal shift software v1

Protein Unfolding Kinetics Monitoring

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To monitor protein unfolding, the fluorescent Protein Thermal Shift™ Dye Kit was used. The unfolding process exposes the hydrophobic region of proteins and results in a significant increase in fluorescence, which monitors the protein-unfolding transition. The thermal shift assay was conducted in the QuantStudio 3 Real-Time-PCR-System (ThermoFisher Scientific, Carlsbad, CA, U.S.A.), initially designed for PCR. The system contains a heating/cooling device for accurate temperature control and a charge-coupled device (CCD) detector for simultaneous imaging of the fluorescence changes in the microplate wells. The final concentration of InsP₃Kinase-A wildtype (wt) and mutants was 6.7 µM, and the final compound concentration was 20 μM. The plate was heated from 25 to 99°C with a heating rate of 0.05°C/s. The fluorescence intensity was measured with Ex/Em: 580/623 nm. The fluorescence imaging data from the CCD detector were analyzed using the Protein Thermal Shift™ Software v1.4 (ThermoFisher Scientific, Carlsbad, CA), and derivative Tm values were obtained.

Paraschiakos T., Flat W., Chen Y., Kirchmair J, & Windhorst S. (2021). Mechanism of BIP-4 mediated inhibition of InsP3Kinase-A. Bioscience Reports, 41(7), BSR20211259.

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Thermal Stability Profiling of Ubiquitin Variants

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10 µl of the wild-type or I3C mutant ubiquitin (500 µM) was mixed with 10 µl of 10x SYPRO Orange Protein Gel Stain (Thermo Fisher) in a MicroAmp Fast 96-Well Reaction Plate (Thermo Fisher). The plate was covered with Optical Adhesive Covers (Thermo Fisher). The fluorescence (excitation at 470 nm and emission at 586.5 nm) was followed using a QuantStudio™ 7 Flex Real-Time PCR System (Thermo Fisher). The 96-well plate was incubated at 25°C for 2 min and then heated up to 99°C at 0.05°C/s, followed by a 2-min incubation at 99°C. Raw data were processed with Protein Thermal Shift Software v1.4 (Thermo Fisher) and fit with the Boltzmann function. The melting temperature (Tm) was calculated from the point of half maximal fluorescence change. See Table S3 for the raw data and the corresponding Boltzmann-fit curves.

Ji Z., Li H., Peterle D., Paulo J.A., Ficarro S.B., Wales T.E., Marto J.A., Gygi S.P., Engen J.R, & Rapoport T.A. (2021). Translocation of polyubiquitinated protein substrates by the hexameric Cdc48 ATPase. Molecular cell, 82(3), 570-584.e8.

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Protein Thermal Shift Assay

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A thermal shift assay using SYPRO orange dye (Thermo Fisher Scientific, Waltham, MA, USA) was performed in 96‐well PCR plates on an Applied Biosystems StepOnePlus real‐time PCR instrument (Thermo Fisher Scientific) in the 25–99 °C range in 1 °C·min⁻¹ increments. The proteins (0.6 mg·mL⁻¹) were added to buffer E containing 100× SYPRO orange dye in a total volume of 20 μL. The melting temperature (T_m) was determined using protein thermal shift software v1.4 (Thermo Fisher Scientific).

Hoang T., Jeong C., Jang S, & Lee C. (2023). Tyr76 is essential for the cold adaptation of a class II glutaredoxin 4 with a heat‐labile structure from the Arctic bacterium Sphingomonas sp.. FEBS Open Bio, 13(3), 500-510.

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Thermal Stability of hCEACAM1 Variants

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Differential scanning fluorimetry was performed using a QuantStudio 6 (Life Technologies) RT-PCR instrument with the excitation and emission wavelengths set to 587 and 607 nm, respectively. Assay buffer was 10 mM HEPES pH 7.4, 150 mM NaCl. For thermal stability measurements, the temperature scan rate was fixed at 1 °C/min. Protein concentration was uniform at 25 μM among the hCEACAM1 WT and mutant samples and SYPRO orange (Invitrogen) concentration was consistent at 5×. The temperature range spanned 20 °C to 95 °C. Data collection was performed by Quant Studio Real-Time PCR Software (Life Technologies) on triplicate samples and analyzed by Protein Thermal Shift Software v1.4 (ThermoFisher). Melting point temperature (T_m) was calculated for each protein samples through computation of a temperature derivative for each respective melting curve that was then processed with a peak fitting algorithm, applying a sigmoidal baseline and fitting the peak to determine the T_m and its standard error.

Gandhi A.K., Sun Z.Y., Kim W.M., Huang Y.H., Kondo Y., Bonsor D.A., Sundberg E.J., Wagner G., Kuchroo V.K., Petsko G.A, & Blumberg R.S. (2021). Structural basis of the dynamic human CEACAM1 monomer-dimer equilibrium. Communications Biology, 4, 360.

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Thermal Stability of Spike Variants

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DSF was used to determine the melting curve of purified spike variants. All samples are formulated in DPBS at pH 7.8. The protein concentration was slightly different from one sample to another and ranged from 1 to 4 µM. A volume of 15 µl of each sample was placed in a 96-well qPCR plate (Applied Biosystems, Cat# N8010560) to which was added 5 µl of a Sypro Orange (Invitrogen, Cat# S6650), pre-diluted 125-fold from the manufacturer’s stock solution with DPBS. The samples were mixed by pipetting up and down after which the plate was sealed with an optical adhesive cover (Applied Biosystems, Cat# 4311971) and centrifuged for 5 min at 1000g. Thermal melt curves were recorded using the qPCR instrument QuantStudio7 with a thermal gradient ramp from 25 to 95 °C at 0.9 °C/min. Data was analyzed using the Protein Thermal Shift software (v1.4) from Thermo Fisher.

Stuible M., Schrag J.D., Sheff J., Zoubchenok D., Lord-Dufour S., Cass B., L’Abbé D., Pelletier A., Rossotti M.A., Tanha J., Gervais C., Maurice R., El Bakkouri M., Acchione M, & Durocher Y. (2023). Influence of variant-specific mutations, temperature and pH on conformations of a large set of SARS-CoV-2 spike trimer vaccine antigen candidates. Scientific Reports, 13, 16498.

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Protein Thermal Shift Analysis

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Protein thermal shift analysis was conducted using SYPRO orange dye (Thermo Fisher Scientific, Waltham, MA, USA) and an Applied Biosystems StepOnePlus real-time PCR instrument (Thermo Fisher Scientific). The assay involved heating the proteins (3 mg/mL) in buffer D with 25× SYPRO orange dye, with a temperature range of 25–99°C and a heating rate of 1°C/min. The thermal denaturation midpoint (T_m value) was determined using Protein Thermal Shift software v1.4 (Thermo Fisher Scientific).

Tran T.V., Hoang T., Jang S.H, & Lee C. (2023). Unraveling the roles of aromatic cluster side-chain interactions on the structural stability and functional significance of psychrophilic Sphingomonas sp. glutaredoxin 3. PLOS ONE, 18(8), e0290686.

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Thermal Stability of Fusion Proteins

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PQBP5, nucleolin, and fibrillarin His-tag fusion proteins were generated as described above, adjusted to 0.3 μM, and dialyzed against to 20 mM Tris-HCl, pH 7.5, 25 mM NaCl, 1 mM DTT using Slide-A-Lyzer MINI Dialysis Units (#69570, Thermo Fisher Scientific). Each well of a PCR plate (MicroAmp™ Fast Optical 96-Well Reaction Plate, #4346907, Live Technologies, Carlsbad, CA, USA) contained 10 μl of a fusion protein, 2.5 μl of Protein Thermal Shift dye (#4462263, Thermo Fisher Scientific) and 7.5 μl of osmotic buffer, resulting in a final volume of 20 μl. Four types of osmotic buffer were used: hypotonic (246.5 mM NaCl); isotonic (353 mM NaCl); hypertonic (620 mM NaCl); and superhypertonic (886.5 mM NaCl). The plates were sealed with MicroAmp Optical Adhesive Film, and the temperature was increased from 25 °C to 99 °C at a rate of 0.4 °C per minute, during which time the fluorescence in each well was monitored. The results were analyzed using Protein Thermal Shift™ Software v1.4 (#4466037, Thermo Fisher Scientific).

Jin X., Tanaka H., Jin M., Fujita K., Homma H., Inotsume M., Yong H., Umeda K., Kodera N., Ando T, & Okazawa H. (2023). PQBP5/NOL10 maintains and anchors the nucleolus under physiological and osmotic stress conditions. Nature Communications, 14, 9.

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Thermal Shift Assay of IGF2BP2 Protein

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The fluorescence protein thermal shift assay was conducted according to a previous repot^{38 (link)}. The recombinant IGF2BP2 protein was purified from 293T cells using the Mammalian Protein Purification System (Promega). The thermal shift assay was conducted in the QuantStudio 12K Flex Real-Time PCR System (ThermoFisher) using the Protein Thermal Shift kit (ThermoFisher) following the manufacturer’s instructions. Briefly, we used 5 μg purified IGF2BP2 protein in a 20 μl thermal shift reaction with indicated concentration of CWI1-2 or vehicle control. The plate was heated from 25 to 99 °C with a heating rate of 0.05 °C/s in the Real-Time PCR System. The fluorescence intensity was measured with Excitation: x4 (580±10) and Emission: m4 (623±14). The fluorescence Tm values were calculated by Boltzmann fitting using the Protein Thermal Shift™ Software v1.4 (ThermoFisher).

Weng H., Huang F., Yu Z., Chen Z., Prince E., Kang Y., Zhou K., Li W., Hu J., Fu C., Aziz T., Li H., Li J., Yang Y., Han L., Zhang S., Ma Y., Sun M., Wu H., Zhang Z., Wunderlich M., Robinson S., Braas D., Hoeve J.T., Zhang B., Marcucci G., Mulloy J.C., Zhou K., Tao H.F., Deng X., Horne D., Wei M., Huang H, & Chen J. (2022). The m6A Reader IGF2BP2 Regulates Glutamine Metabolism and Represents a Therapeutic Target in Acute Myeloid Leukemia. Cancer cell, 40(12), 1566-1582.e10.

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Thermal Shift Assay for scFv Characterization

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Thermal shift assays were performed as previously described^{53 (link)} with minor modifications. SYPRO Orange dye was firstly mixed with 5 μM final concentration of scFv according to user manual (ThermoFisher Scientific). An optical 96-well reaction plate (Applied Biosystems) loaded with 20 μl reaction mixture per well was mounted onto a real-time PCR instrument (QuantStudio 6 Flex, Applied Biosystems). The mixture was heated up by 0.05 °C/s from 30 to 90 °C, and the fluorescence signal in 570-nm wavelength yielded by the dye was recorded concomitantly. Melting temperature (Tm) for each scFv was determined by fitting fluorescence signal and temperature to the Boltzmann equation using Protein Thermal Shift software v1.3 (ThermoFisher Scientific).

Wang Y., Yuan W., Guo S., Li Q., Chen X., Li C., Liu Q., Sun L., Chen Z., Yuan Z., Luo C., Chen S., Tong S., Nassal M., Wen Y.M, & Wang Y.X. (2022). A 33-residue peptide tag increases solubility and stability of Escherichia coli produced single-chain antibody fragments. Nature Communications, 13, 4614.

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Thermal Stability Profiling of Phenylalanyl-tRNA Synthetases

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These were performed with TgcPheRS and HscPheRS enzymes with BRD7929, BRD2108, mCMY416 by diluting the purified enzyme to a final concentration of 2 μM in a buffer containing 200 mM NaCl, 5 mM MgCl2, 50 mM Tris (pH 7.5), and 2 X SYPRO orange dye (Life Technologies)^{6 (link), 12 (link), 13 (link)}. Each compound at a concentration of 20 μM was individually added to the enzyme solutions and samples were incubated at room temperature for 10min. The solution containing the purified enzymes alone (Apo) and with natural substrates (ATP and L-phenylalanine) were kept as control. The samples were briefly subjected to temperature ramp from 25°C TO 99°C at a rate of 1°C min⁻¹ and the fluorescence signals of SYPRO^® orange dye were monitored using StepOnePlus ^™ quantitative real-time PCR system (Life Technologies). Each melting curve is an average of three measurements and analysis was done using Protein Thermal shift software (v1.3, Thermofisher). The derivative Tm was used for analysis.

Ence C.C., Uddin T., Borrel J., Mittal P., Xie H., Zoller J., Sharma A., Comer E., Schreiber S.L., Melillo B., Sibley L.D, & Chatterjee A.K. (2024). Bicyclic pyrrolidine inhibitors of Toxoplasma gondii phenylalanine t-RNA synthetase with antiparasitic potency in vitro and brain exposure. bioRxiv.

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