protein thermal shift starter kit, by Thermo Fisher Scientific - Product details

1

Thermal Shift Assay for Protein Folding

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Thermal shift assays were performed using a Real-Time PCR Detection System (StepOne Real-Time PCR System; Applied biotechnology, CA, USA) with a temperature increment of 0.2 °C and a temperature range of 25–95 °C. A total of 25 μL of mixtures containing 2.5 μL of protein dye (Protein Thermal Shift Starter Kit; Life Technologies, Carlsbad, CA, USA; diluted from 5000x concentrate stock), 10μL of reaction buffer (Protein Thermal Shift Starter Kit) and 12.5 μL of protein (at a concentration of 0.5 mM), was mixed on ice in a 96-well plate. The mid-denaturation temperatures (Tm) that measure protein folding and unfolding transitions were estimated using the device with the following equation [34 (link), 35 ]:

\begin{matrix} I = (A + \frac{B - A}{1 + e^{(T_{m} - T) / C}}) \end{matrix}

where I is the fluorescence intensity at temperature T, A and B are the pre-transitional and post-transitional fluorescence intensities, respectively, and C is a slope factor.

Tan F, & Xu J. (2022). Validation of the solution structure of dimerization domain of PRC1. PLoS ONE, 17(8), e0270572.

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Thermal Shift Assay for Protein Folding

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Thermal shift assays were performed using a Real-Time PCR Detection System (StepOne Real-Time PCR System, Applied Biotechnology, San Luis Obispo, CA, USA) with a temperature increment of 0.2

^{°}

C and a temperature range of 25–95

^{°}

C. A total of 25

μ

L of mixtures containing 2.5

μ

L of protein dye (Protein Thermal Shift Starter Kit, Life Technologies, Carlsbad, CA, USA; diluted from 5000× g concentrate stock), 10

μ

L of reaction buffer (Protein Thermal Shift Starter Kit) and 12.5

μ

L of protein (at a concentration of 0.5 mM), was mixed on ice in a 96-well plate. The mid-denaturation temperatures (Tm) that measure protein folding and unfolding transitions were estimated using the device with the following equation [51 (link),52 ,53 (link)]:

I = (A + \frac{B - A}{1 + e (T_{m} - T) / C})

where I is the fluorescence intensity at temperature T, A and B are the pre-transitional and post-transitional fluorescence intensities, respectively, and C is a slope factor [54 (link)].

Tan F, & Xu J. (2022). N-Terminus-Mediated Solution Structure of Dimerization Domain of PRC1. Current Issues in Molecular Biology, 44(4), 1626-1645.

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3

Protein Thermal Stability Assay

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Protein stability was measured using the protein thermal shift starter kit (Thermo Fisher Scientific, Waltham, MA, USA). A 20-µL sample mixture of protein, thermal shift dye, and buffer was tested. The fluorescent signal was detected as a function of temperature using a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Raw data were retrieved using the Transcriptic webapp, and melt curves were generated by plotting the first derivative of the fluorescent signal as a function of temperature.

Bhat E.A., Kim C.M., Kim S, & Park H.H. (2018). In Vitro Inhibitory Mechanism Effect of TRAIP on the Function of TRAF2 Revealed by Characterization of Interaction Domains. International Journal of Molecular Sciences, 19(8), 2457.

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4

Thermal Shift Assay of BOK and BAK Proteins

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TSAs were performed by using the Protein Thermal Shift Starter Kit
(ThermoFisher), according to the manufacturer’s protocol, using 0.25
mg/mL purified BOK and BAK. Briefly, for each protein sample, a 90 μL
MasterMix was prepared by using the buffer and the fluorescent dye ROX,
vortexed at low speed, and 4 aliquots of 20 μL each were dispensed in
4 individual wells of Microamp Fast Optical 96-well reaction plates
(ThermoFisher) for analysis in an Applied Biosystems 7500 thermal cycler
(ThermoFisher). The temperature gradient was 25°C–99°C
at a rate of 1°C/min. Data were analyzed with Protein Thermal Shift
Software v1.3 (ThermoFisher) to determine the melting temperature, using the
first derivative and Boltzmann equation non-linear regression. Dye-only
controls were included in each plate. Experiments were performed at least 2
times in quadruplicates for each protein sample at the 2 protein
concentrations. More recently, TSA assays were performed in 20 mM NaOAc pH
5.0 or 20 mM HEPES pH 6.8 and 150 mM NaCl, and 384-well plates in an Applied
Biosystems 7900HT Fast Real-Time PCR System (ThermoFisher). Final data
analysis and presentation were done in Microsoft Excel and GraphPad Prism v7
(GraphPad Software).

Zheng J.H., Grace C.R., Guibao C.D., McNamara D.E., Llambi F., Wang Y.M., Chen T, & Moldoveanu T. (2018). Intrinsic Instability of BOK Enables Membrane Permeabilization in Apoptosis. Cell reports, 23(7), 2083-2094.e6.

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5

Thermal Shift Assay for THP-1 Cells

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GTx1-15 was obtained from Alomone Labs (Jerusalem, Israel). Human plasma was purchased from KOJIN BIO (Saitama, Japan). The protein thermal shift starter kit was from Thermo Fisher Scientific (Tokyo, Japan). RPMI-1640 with L-glutamine and phenol red, sodium hydrogen carbonate, and Tris-HCl were purchased from Wako Chemicals (Osaka, Japan). A human monocytic leukemia cell line, THP-1 (No. JCRB0112), was from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). Penicillin-streptomycin solution was obtained from Life Technologies Japan Ltd. (Tokyo, Japan). HyClone fetal bovine serum was purchased from GE Healthcare Japan (Tokyo, Japan). Premix WST-1 reagent was purchased from Takara Bio (Shiga, Japan).

, & Kimura T. (2021). Stability and Safety of Inhibitor Cystine Knot Peptide, GTx1-15, from the Tarantula Spider Grammostola rosea. Toxins, 13(9), 621.

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6

Thermal Shift Assay of BOK and BAK Proteins

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TSAs were performed by using the Protein Thermal Shift Starter Kit
(ThermoFisher), according to the manufacturer’s protocol, using 0.25
mg/mL purified BOK and BAK. Briefly, for each protein sample, a 90 μL
MasterMix was prepared by using the buffer and the fluorescent dye ROX,
vortexed at low speed, and 4 aliquots of 20 μL each were dispensed in
4 individual wells of Microamp Fast Optical 96-well reaction plates
(ThermoFisher) for analysis in an Applied Biosystems 7500 thermal cycler
(ThermoFisher). The temperature gradient was 25°C–99°C
at a rate of 1°C/min. Data were analyzed with Protein Thermal Shift
Software v1.3 (ThermoFisher) to determine the melting temperature, using the
first derivative and Boltzmann equation non-linear regression. Dye-only
controls were included in each plate. Experiments were performed at least 2
times in quadruplicates for each protein sample at the 2 protein
concentrations. More recently, TSA assays were performed in 20 mM NaOAc pH
5.0 or 20 mM HEPES pH 6.8 and 150 mM NaCl, and 384-well plates in an Applied
Biosystems 7900HT Fast Real-Time PCR System (ThermoFisher). Final data
analysis and presentation were done in Microsoft Excel and GraphPad Prism v7
(GraphPad Software).

Zheng J.H., Grace C.R., Guibao C.D., McNamara D.E., Llambi F., Wang Y.M., Chen T, & Moldoveanu T. (2018). Intrinsic Instability of BOK Enables Membrane Permeabilization in Apoptosis. Cell reports, 23(7), 2083-2094.e6.

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7

Protein Thermal Stability Assay

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The stability of wild-type and mutant proteins was assessed using the QuantStudio™ 3 Real-Time PCR System and the Protein Thermal Shift™ Starter Kit (Applied Biosystems), as per manufacturers’ instructions. The fluorescence of the solution was then measured over a range of temperatures (23–99 °C), and the resultant data were analyzed using the Protein Thermal Shift™ Software v1.4 (Applied Biosystems).

Blake K.S., Kumar H., Loganathan A., Williford E.E., Diorio-Toth L., Xue Y.P., Tang W.K., Campbell T.P., Chong D.D., Angtuaco S., Wencewicz T.A., Tolia N.H, & Dantas G. (2024). Sequence-structure-function characterization of the emerging tetracycline destructase family of antibiotic resistance enzymes. Communications Biology, 7, 336.

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8

Protein Thermal Shift Assay Protocol

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All thermal shift assays were performed
with an Applied Biosystems Protein Thermal Shift Starter Kit (Cat.
No. 4462263). Replicates containing 1 μM tES/tES-rLuc in 1×
PBS were mixed with a 1× dye provided in the kit. Reactions were
carried out in 384-well PCR plates (Thermo Scientific), which were
sealed with adhesive sealing sheets (AB-0558, Thermo Scientific).
The plates were analyzed on an Applied ViiA 7 Real-Time PCR system
at temperatures ranging from 25 to 99.9 °C at a ramp rate of
0.05 °C·s^–1.

Sadeghi S., Masurkar N.D., Vallerinteavide Mavelli G., Deshpande S., Kok Yong Tan W., Yee S., Kang S.A., Lim Y.P., Kai-Hua Chow E, & Drum C.L. (2022). Bioorthogonal Catalysis for Treatment of Solid Tumors Using Thermostable, Self-Assembling, Single Enzyme Nanoparticles and Natural Product Conversion with Indole-3-acetic Acid. ACS Nano, 16(7), 10292-10301.

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9

Thermal Stability Shift Assay for Recombinant Proteins

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Protein thermal stability shift assay was performed as per the manufacturer’s protocols (Protein thermal Shift starter kit, Cat no: 4462263, Applied Biosystems). In brief, all the reactions were set up in final volumes of 20 μl in 96-well plates. In each well, 1 μg of purified recombinant protein was added with 5 μl of thermal stability buffer and 2.5 μl of 8X dye with varying amounts of NAD⁺ (0, 1, 2 and 4 mM). For each set, samples were made in quadruple and at least three biologically independent experiments were conducted. Temperature gradient was set in the range of 25°C to 99°C with a 0.05°C/s ramp rate using real-time PCR system (Applied Biosystems). Melting temperature (Tm) was calculated by using Protein Thermal Shift Software 1x (Applied Biosystems).

Manna D., Lentz C.S., Ehrenkaufer G.M., Suresh S., Bhat A, & Singh U. (2018). An NAD+-dependent novel transcription factor controls stage conversion in Entamoeba. eLife, 7, e37912.

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Protein thermal shift starter kit

Lab products found in correlation

9 protocols using protein thermal shift starter kit

Thermal Shift Assay for Protein Folding

Thermal Shift Assay for Protein Folding

Protein Thermal Stability Assay

Thermal Shift Assay of BOK and BAK Proteins

Thermal Shift Assay for THP-1 Cells

Thermal Shift Assay of BOK and BAK Proteins

Protein Thermal Stability Assay

Protein Thermal Shift Assay Protocol

Thermal Stability Shift Assay for Recombinant Proteins

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