Sypro orange

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, United Kingdom, Spain, Canada, Italy, Sweden, Austria

SYPRO Orange is a fluorescent dye used in protein analysis. It binds to proteins and emits a fluorescent signal that can be detected and quantified. The dye is commonly used in techniques such as thermal shift assays and protein folding studies to monitor protein stability and unfolding.

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Lab products found in correlation

691 protocols using sypro orange

Protein Thermal Stability Assay

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Thermal melting experiments were carried out using a QuantStudio 6 and 7 Flex Real-Time PCR Systems (Life) as described previously (Niesen et al., 2007 (link)). The buffer is 50 mM HEPES, 150 mM KCl, 10 mM MgCl₂, and 5% glycerol at pH 8.0. SYPRO Orange (Thermo Fisher) was added as a fluorescence probe at a dilution of 1:1000. 10 μL of protein mixed with SYPRO Orange (Thermo Fisher) solution (1:1000, 50 mM HEPES, 150 mM KCl, 10 mM MgCl₂, and 5% glycerol at pH 8.0) to a final concentration of 10 μM were assayed in 384-well plates (Life). Excitation and emission filters for SYPRO-Orange dye were 465 nm and 590 nm, respectively. The temperature was increased by 0.9°C per minute from 25°C to 96°C. The inflection point of the transition curve (Tm) is calculated using protein thermal shift software v1.2 (Thermo Fisher).

Ma X., Zhu Y., Lu J., Xie J., Li C., Shin W.S., Qiang J., Liu J., Dou S., Xiao Y., Wang C., Jia C., Long H., Yang J., Fang Y., Jiang L., Zhang Y., Zhang S., Zhai R.G., Liu C, & Li D. (2020). Nicotinamide mononucleotide adenylyltransferase uses its NAD+ substrate-binding site to chaperone phosphorylated Tau. eLife, 9, e51859.

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Thermally Induced Protein Unfolding Assays

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Thermally induced protein unfolding assays were used to determine the melting temperatures (Tm) of adhirons. The optimal signal-to-noise ratio between adhiron and dye (0.1 μg/μL of protein sample and 5x of SYPRO orange, Thermo Fisher) was identified in preliminary tests performed using different concentration combinations. Adhirons were diluted in buffer A (50 mM Hepes pH 6.5) or B (50 mM PBS pH 7.4) according to pI and further mixed with the dye (SYPRO orange, Thermo Fisher Scientific); 50 μL/sample was added to MicroAmp 96-Thermowell PCR plates (Thermo Fisher). The effect of additives (100 mM NaCl, 5 mM DTT, or 5 mM EDTA) was also tested. Reactions were carried out in a Light-Cycler 480 II real-time qPCR (Roche—Basel, Switzerland) with a temperature gradient ranging from 25 °C to 95 °C and an amp rate of 0.01 °C/s. Fluorescence was detected using an excitation wavelength of 465 nm, an emission of 580 nm with continuous acquisition mode, and recording 100 acquisitions per 1 °C. Melting temperatures were determined with the Origin 8.1 software (OriginLab Corporation—Northampton, MA, USA) using the Boltzmann function.

D’Ercole C., De March M., Veggiani G., Oloketuyi S., Svigelj R, & de Marco A. (2023). Biological Applications of Synthetic Binders Isolated from a Conceptually New Adhiron Library. Biomolecules, 13(10), 1533.

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Optimizing LbSOD Thermal Stability

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Conditions were optimized to LbSOD: protein concentration, buffer, and DMSO concentration. The protein concentration was evaluated (1–5 µM) in the presence of SYPRO Orange^® (Thermofisher, Waltham, MA, USA) (1:100 dilution) in ultrapure water qsp 20 µL. Then, the buffers (sodium citrate, sodium phosphate, tris-HCl, and glycine) and pHs (4–9) were evaluated at a final concentration of 50 mM for each buffer, SYPRO Orange^® (1: 100 dilution) and LbSOD. Finally, the influence of DMSO (2.5, 5, 10% v/v) on the thermal stability of the protein was investigated.

Brito C.C., da Silva H.V., Brondani D.J., de Faria A.R., Ximenes R.M., da Silva I.M., de Albuquerque J.F, & Castilho M.S. (2018). Synthesis and biological evaluation of thiazole derivatives as LbSOD inhibitors. Journal of Enzyme Inhibition and Medicinal Chemistry, 34(1), 333-342.

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Thermal Stability Assay of QseB and QseBN

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QseB and QseBN were dialyzed into reaction buffer containing 20 mM MES pH 7.0, 100 mM NaCl, and 10% v/v glycerol. AGL compounds were dissolved in 100% PEG 400 to a final concentration of 1 mM. For QseB, reactions were carried out using final concentrations of 5 µM QseB, 25 µM compound, 10% v/v PEG 400, and 10× SYPRO orange (ThermoFisher Scientific). Samples were prepared in three technical replicates on a CFX384 Touch Real-Time PCR Detection System (BioRad). Samples were heated from 25° to 95°C in 0.5°C increments, holding for 30 sec at each step. Fluorescence was detected using the default HEX wavelengths. Data was fit to a Boltzmann curve using SigmaPlot. For QseBN, reactions were carried out using final concentrations of 100 µM QseBN, 100 to 400 µM compound, 10% PEG 400, and 8× SYPRO orange (ThermoFisher Scientific). Samples were prepared in technical replicates on a QuantStudio 3 (Applied Biosystems). Samples were headed from 25° to 99° C in 0.05°C/s. Fluorescence was detected using ROX as the reporter. Data was analyzed using Protein Thermal Shift Software (Applied Biosystems) and fit to a Boltzmann curve. Assays were repeated in triplicate.

Milton M.E., Allen C.L., Feldmann E.A., Bobay B.G., Jung D.K., Stephens M.D., Melander R.J., Theisen K.E., Zeng D., Thompson R.J., Melander C, & Cavanagh J. (2017). Structure of the Francisella response regulator QseB receiver domain, and characterization of QseB inhibition by antibiofilm 2-aminoimidazole-based compounds. Molecular microbiology, 106(2), 223-235.

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Thermal Shift Assay for ENPP1 Inhibitors

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Example 2

Materials:

Recombinant Human ENPP-1 Protein (Purified In-House)

Assay Buffer (1 mM CaCl₂, 0.2 mM ZnCl₂, 50 mM Tris, pH 9.0)

5000×SYPRO Orange (ThermoFisher cat #S6651)

384-well PCR Plates

Protocol:

Each drug was prepared as a 1 Ox solution in the assay buffer and SYPRO Orange was diluted to 10× concentration in water. Wells were set up in duplicate in a 384-well PCR plate as follows: 14 μL assay buffer, 2 μL ENPP1 Inhibitor or DMSO, 2 μL (0.5 μg) ENPP1 protein. Each well was mixed and incubated on ice for 5 minutes. Post incubation, 2 μL of SYPRO Orange was mixed into each well and followed by a gentle centrifugation. The protein melt reaction was run using ViiA7 software with temperatures beginning at 25° C. and increasing by 0.05° C./s to the maximum temperature of 99° C.

Thermal Shift

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US11591313B2. Inhibitors of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and methods of use thereof (2023-02-28). Stingray Therapeutics, Inc. [US]. Inventors: Srinivas Rao Kasibhatla [US], Raman Kumar Kalakuntla [IN], Alexis Weston [US], Trason Thode [US], Sunil Sharma [US], Mohan R. Kaadige [US].

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Differential Scanning Fluorimetry for Protein Thermal Stability

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For differential scanning fluorimetry, we mixed protein and dye solution in 96 well plates to a total volume of 20 µL per well. 4 µL mCes2c per well (1.3 mg/mL) were mixed with 14 µL buffer B, 2 µL of 50X SYPRO Orange (Thermo Fisher Scientific) per well were added to reach the final concentration of 5X for SYPRO Orange. The temperature was increased from 20 °C to 95 °C with increments of 0.5 °C every 30 s. The melting temperature (T_M) was calculated from the first derivate of the fluorescence signal vs. the temperature. Experiments were performed as triplicates.

Eisner H., Riegler-Berket L., Gamez C.F., Sagmeister T., Chalhoub G., Darnhofer B., Jazleena P.J., Birner-Gruenberger R., Pavkov-Keller T., Haemmerle G., Schoiswohl G, & Oberer M. (2022). The Crystal Structure of Mouse Ces2c, a Potential Ortholog of Human CES2, Shows Structural Similarities in Substrate Regulation and Product Release to Human CES1. International Journal of Molecular Sciences, 23(21), 13101.

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Thermal Unfolding Analysis of Proteins

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SYPRO Orange dye
has a higher fluorescence intensity when bound to exposed hydrophobic
functional groups of proteins, and thus, it provides a probe for proteins’
thermal unfolding.^{68 (link)−70 (link)} Differential scanning fluorimetry experiments were
carried out in a QuantStudio 6 Flex real-time PCR system (ThermoFisher),
using a 384-well white round-bottom plate, 20–30 μL reaction
volume, excitation wavelength of 580 nm, and emission detected using
the ROX filter setting (623 nm). Samples were prepared in PBS with
5 μM protein, 5× SYPRO Orange (ThermoFisher), and 1.1%
DMSO with or without 100 μM Stitch-3. The scFv melting curve
was collected from 25 to 95 °C (0.5 °C/min), and data were
analyzed with PRISM 6 (GraphPad) using the Gibbs–Helmholtz
equation^{71 (link)} to determine the Tm values.
Samples were run in 8 replicates.

Khowsathit J., Bazzoli A., Cheng H, & Karanicolas J. (2020). Computational Design of an Allosteric Antibody Switch by Deletion and Rescue of a Complex Structural Constellation. ACS Central Science, 6(3), 390-403.

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Differential Scanning Fluorimetry of MA Protein

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A protocol for Differential Scanning Fluorimetry (DSF) was employed according to the previous procedures^{42 (link),43 (link)}. SYPRO® Orange (Thermo Fisher Scientific, Massachusetts, United States of America) solution (× 5000) was diluted by 50 times with a buffer containing 50 mM MOPS-NaOH (pH 7.0), 50 mM NaCl (100 × SYPRO® Orange solution). A buffer solution containing purified His10-tagged MA protein was exchanged to the 50 mM MOPS-NaOH, 50 mM NaCl buffer (pH 7.0) with ultrafiltration using Amicon® Ultra-15 Centrifugal Filter Unit (10 kDa MWCO) (Merck, Darmstadt, Germany). With the prepared 100 × SYPRO® Orange solution and the protein solution, 50 μl of a reaction mixture were prepared to be 5.4 µM MA-His10 protein, 5 × SYPRO® Orange in presence of or in absence of the indicated concentration of compounds to evaluate the contribution of IP6 to the thermostability of MA protein, or 32.4 µM IP6 for MA point-mutant analysis. The temperature of the reaction system was increased 0.5 °C per 30 s from 20 to 95 °C stepwise, and fluorescence intensity at each temperature was measured by Single-Color Real-Time PCR Detection System, MyiQ (Bio-Rad, California, United States of America), and analyzed with iQ5 (Bio-Rad).

Ciftci H., Tateishi H., Koiwai K., Koga R., Anraku K., Monde K., Dağ Ç., Destan E., Yuksel B., Ayan E., Yildirim G., Yigin M., Ertem F.B., Shafiei A., Guven O., Besler S.O., Sierra R.G., Yoon C.H., Su Z., Liang M., Acar B., Haliloglu T., Otsuka M., Yumoto F., Fujita M., Senda T, & DeMirci H. (2021). Structural insight into host plasma membrane association and assembly of HIV-1 matrix protein. Scientific Reports, 11, 15819.

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Differential Scanning Fluorimetry of Obscurin

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Purified obscurin Ig58, Ig58_R4344Q, Ig58-Ig59, Ig58-Ig59_R4344Q, Ig58-Ig59_R4444W, Ig58-Ig59_A4484T, and Ig58-Ig59_{R4344Q, A4484T} were mixed with SYPRO orange (ThermoFisher) to a final concentration of 20 μM and 20X, respectively. 20 μl of each protein-SYPRO orange mix was pipetted into 4–5 wells of a white 96-well PCR plate (ThermoFisher) covered with optical PCR seals (Bio-Rad) and DSF was run according to Niesen et al.(16 (link)); briefly, the plate was loaded into a Stratagene Mx3005p and heated from 25 to 96°C, 1^oC per minute, and fluorescent emission of SYPRO orange was measured at each degree °C at 610 nm following excitation at 492 nm. The data relating to the unfolding of the protein were automatically identified using a Microsoft Excel spreadsheet from Niesen et al., with the melting temperature, Tm, calculated in GraphPad Prism (v8.3) by fitting the curve to the Boltzman Equation , where LL and UL refer to the lower and upper fluorescent intensities, Tm is the melting temperature, Y is the fluorescence at x, x is the temperature and a is the slope of the curve at Tm.

Fukuzawa A., Koch D., Grover S., Rees M, & Gautel M. (2021). When is an obscurin variant pathogenic? The impact of Arg4344Gln and Arg4444Trp variants on protein–protein interactions and protein stability. Human Molecular Genetics, 30(12), 1131-1141.

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Differential Scanning Fluorimetry of MA Protein

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A protocol for Differential Scanning Fluorimetry (DSF) was employed according to the previous procedures 42, 43 . SYPRO ® Orange (Thermo Fisher Scientific, Massachusetts, United States of America) solution (× 5000) was diluted by 50 times with a buffer containing 50 mM MOPS-NaOH (pH 7.0), 50 mM NaCl (100 × SYPRO ® Orange solution). A buffer solution containing purified His10tagged MA protein was exchanged to the 50 mM MOPS-NaOH, 50 mM NaCl buffer (pH 7.0) with ultrafiltration using Amicon ® Ultra-15 Centrifugal Filter Unit (10 kDa MWCO) (Merck, Darmstadt, Germany). With the prepared 100 × SYPRO ® Orange solution and the protein solution, 50 μl of a reaction mixture were prepared to be 5.4 µM MA-His10 protein, 5 × SYPRO ® Orange in presence of or in absence of the indicated concentration of compounds to evaluate the contribution of IP6 to the thermostability of MA protein, or 32.4 µM IP6 for MA point-mutant analysis. The temperature of the reaction system was increased 0.5°C per 30 sec from 20 to 95°C stepwise, and fluorescence intensity at each temperature was measured by Single-Color Real-Time PCR Detection System, MyiQ (Bio-Rad, California, United States of America), and analyzed with iQ5 (Bio-Rad).

Ciftci H.I., Tateishi H., Koiwai K., Koga R., Anraku K., Monde K., Dağ Ç., Destan E., Yuksel B., Ayan E., Yildirim G., Yigin M., Ertem F.B., Shafiei A., Güven Ö., Besler S.O., Sierra R.G., Yoon C.H., Su Z., Li M.J., Acar B., Haliloğlu T., Otsuka M., Yumoto F., Fujita M., Senda T., & DeMi̇rci̇ H. (2021). Structural Insight Into Host Plasma Membrane Association and Assembly of HIV-1 Matrix Protein.

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