Sypro orange

Manufactured by Bio-Rad

Sourced in United States

SYPRO Orange is a fluorescent dye used for the detection of proteins in polyacrylamide gels. It binds to proteins and emits a fluorescent signal upon excitation, allowing for the visualization and quantification of protein bands.

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Lab products found in correlation

Sypro orange, by Thermo Fisher Scientific (15 mentions) Sypro orange, by Merck Group (6 mentions) Cfx manager 3, by Bio-Rad (6 mentions) Cfx connect real time pcr detection system, by Bio-Rad (4 mentions) Cfx96 qrt pcr thermocycler, by Bio-Rad (4 mentions) C1000 touch thermal cycler, by Bio-Rad (3 mentions) Cfx96 real time system, by Bio-Rad (3 mentions) Optically clear pcr tube, by Avantor (2 mentions) Sypro orange dye, by Thermo Fisher Scientific (2 mentions) C1000 thermocycler with cfx96 real time system, by Bio-Rad (2 mentions) Cfx96 touch real time pcr detection system, by Bio-Rad (2 mentions) 96 well thin wall pcr plate, by Bio-Rad (2 mentions) Iq5 multicolor real time pcr detection system, by Bio-Rad (1 mentions) Hitrap desalt column, by GE Healthcare (1 mentions) Hitrap, by Cytiva (1 mentions) Miniopticon real time pcr detection system, by Bio-Rad (1 mentions) Originpro 2020b, by OriginLab (1 mentions) C1000 thermocycler, by Bio-Rad (1 mentions) Opticon 2 real time pcr detector, by Bio-Rad (1 mentions) Cfx384 touch, by Bio-Rad (1 mentions)

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Sypro Orange dye (6 citations)

28 protocols using sypro orange

Thermal Unfolding of Lymphostatin Protein

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The temperature-induced unfolding of lymphostatin was followed using the environmentally sensitive dye SYPRO Orange (Invitrogen). SYPRO Orange shows an increase in fluorescence correlated with the exposure of hydrophobic residues upon unfolding.44 (link), 45 (link) The fluorescence of WT rLifA and rLifA^C1480A (both 0.1 µM) in 5 × SYPRO Orange was measured between 15 and 70 °C at 0.5 °C increments every 30 s (Bio-Rad IQ5 Multicolor Real-Time PCR Detection System). The proteins were exchanged into Assay Buffer prior to analysis using a HiTrap desalt column (GE Healthcare) at a flow rate of 4 mL/min. Relative fluorescence units (RFU) for each sample were plotted against temperature and the unfolding transition temperature (T_m) of each protein was calculated from the steepest part of the melting curve. Experiments were repeated in triplicate.

Bease A.G., Blackburn E.A., Chintoan-Uta C., Webb S., Cassady-Cain R.L, & Stevens M.P. (2021). Activity of Lymphostatin, A Lymphocyte Inhibitory Virulence Factor of Pathogenic Escherichia coli, is Dependent on a Cysteine Protease Motif. Journal of Molecular Biology, 433(19), 167200.

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Thermal Stability Analysis of SARS-CoV-2 Spike Protein

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200 ng/μL of purified S protein and 5× SYPRO orange (Thermo Fisher Scientific) were added into 20 mM Tris-HCl pH 8.0, 150 mM NaCl in optically clear tubes. SYPRO orange fluorescence intensity in relative fluorescence units (RFU) was measured over temperatures ranging from 10 °C to 95 °C using a CFX Connect Real-Time PCR Detection System (Bio-Rad). Melting temperature (T_m) was calculated as the temperature at which the first derivative of fluorescence intensity with respect to temperature,

- \frac{d (RFU)}{dT}

, was minimum.

Tan T.J., Mou Z., Lei R., Ouyang W.O., Yuan M., Song G., Andrabi R., Wilson I.A., Kieffer C., Dai X., Matreyek K.A, & Wu N.C. (2022). High-throughput identification of prefusion-stabilizing mutations in SARS-CoV-2 spike. bioRxiv.

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Phos-Tag Analysis of KaiC Phosphorylation

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Samples of each KaiC variant were freshly prepared in KaiC buffer and mixed with KaiA to generate a final concentration of 1.5 μM KaiA and 3.4 μM KaiC. Samples were incubated at 30 °C for 24 h, at which point 50 μl was removed and quenched with 10 μl of 6× SDS before incubating at 95 °C for 5 min.
Phos-Tag acrylamide gels were prepared with a resolving gel consisting of 10% (w/v) acrylamide (29:1 acrylamide:bis-acrylamide) containing Tris-HCl pH 8.8 supplemented with 50 μM Phos-tag reagent and 100 μM Mn²⁺. Gels were run at constant 25 mA with a 165 V limit for 35 min before loading any samples into the gel. Samples were loaded and run at room temperature with the same mA and voltage parameters as stated above for 170–190 min. Gels were stained using SYPRO Orange following the Bio-Rad protocol and density was analyzed using ImageJ software^{63 (link)}. Error was estimated as the standard deviation from n = 3 replicate measurements analyzed separately.

Swan J.A., Sandate C.R., Chavan A.G., Freeberg A.M., Etwaru D., Ernst D.C., Palacios J.G., Golden S.S., LiWang A., Lander G.C, & Partch C.L. (2022). Coupling of distant ATPase domains in the circadian clock protein KaiC. Nature structural & molecular biology, 29(8), 759-766.

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Thermal Stability Assay for Proteins

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5 µg protein was mixed with 5

\times

SYPRO orange (Thermo Fisher Scientific) in 20 mM Tris-HCl pH 8.0, 100 mM NaCl, and 10 mM CaCl₂ at a final volume of 25 µL. The sample mixture was then transferred into an optically-clear PCR tube (VWR). SYPRO orange fluorescence data in relative fluorescence unit (RFU) was collected from 10 °C to 95 °C using a CFX Connect Real-Time PCR Detection System (Bio-Rad). The temperature corresponding to the lowest point of the first derivative −d(RFU)/dT was determined to be the T_m.

Lei R., Tan T.J., Hernandez Garcia A., Wang Y., Diefenbacher M., Teo C., Gopan G., Tavakoli Dargani Z., Teo Q.W., Graham C.S., Brooke C.B., Nair S.K, & Wu N.C. (2022). Prevalence and mechanisms of evolutionary contingency in human influenza H3N2 neuraminidase. Nature Communications, 13, 6443.

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Measuring Thermal Stability of Protein Variants

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The melting temperatures of DJ-1 variants were determined using the differential scanning fluorimetry assay (Pantoliano et al. 2001 (link)). Proteins were diluted to 1–5 mg ml⁻¹ in storage buffer (25 mM HEPES pH 7.5, 100 mM KCl, 2 mM DTT). Sypro Orange (Life Technologies, Invitrogen) was diluted to 50, 100, 250, 500× in storage buffer and added to the samples. Samples were heated from 20–95°C at a rate of 2°C min⁻¹ while the fluorescence emission of Sypro Orange was monitored at 575 nm in 8-strip PCR tubes with optically clear caps (Bio-Rad) in a Bio-Rad iCyclerQ real-time PCR. The data were plotted as the first derivative of fluorescence as a function of temperature, where the maximum of this function was taken as the melting temperature (T_m).

Prahlad J., Hauser D.N., Milkovic N.M., Cookson M.R, & Wilson M.A. (2014). Use of cysteine-reactive crosslinkers to probe conformational flexibility of human DJ-1 demonstrates that Glu18 mutations are dimers. Journal of neurochemistry, 130(6), 839-853.

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Thermal Stability Analysis of OTC Variants

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Melting temperatures of WT OTC and variants were determined with a Bio-Rad C1000 Touch Thermal Cycler (CFX96 Real-Time System) and Bio-Rad CFX Manager 3.1.1517.0823 software that measures fluorescence due to Sypro Orange binding to hydrophobic regions of the protein as the temperature is increased from 4°C to 100°C in 0.2°C increments with a 10-sec dwell time [47 (link), 48 (link)]. Samples of wild-type OTC and variants, analyzed in triplicate, were prepared in 96-well plates containing 50 mM HEPES, pH 8, 200 mM NaCl, 2 mM DTT, 10x Sypro Orange (Invitrogen) and 10 μM enzyme alone, enzyme with 1 mM carbamoyl phosphate, enzyme with 10 mM ornithine or enzyme with 10 mM citrulline. Melting temperatures (T_m) were obtained from the software as the highest peak of a first derivative plot of the negative rate change in relative fluorescence (RFU) vs. temperature (T).

Ngu L., Winters J.N., Nguyen K., Ramos K.E., DeLateur N.A., Makowski L., Whitford P.C., Ondrechen M.J, & Beuning P.J. (2020). Probing remote residues important for catalysis in Escherichia coli ornithine transcarbamoylase. PLoS ONE, 15(2), e0228487.

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Thermal Shift Assay for hNV-RdRp Inhibition

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Thermofluorimetric (Thermal shift) assays for the evaluation of the hNV-RdRp melting temperature (T_m) in the absence/presence of the inhibitors, were conducted in a MiniOpticon Real Time PCR Detection System (Bio-Rad), using the fluorescent dye Sypro Orange. Solutions of 4 μl of the NV-RdRp domain (final hNV-RdRp concentration 7 μM, final mNV-RdRp concentration 1.6 μM) were diluted in 9.5 μl of its buffer, and mixed with 3.5 μl of Sypro Orange (Sigma) diluted 60×, and 1 μl of 6 or 8 (4 μM final concentration). In control samples the inhibitors were replaced by water. The sample plates were heated from 20 to 90°C with a heating rate of 0.2 °C/min. Fluorescence intensities were measured within excitation/emission ranges of 470–505 nm and 540–700 nm, respectively.

Croci R., Pezzullo M., Tarantino D., Milani M., Tsay S.C., Sureshbabu R., Tsai Y.J., Mastrangelo E., Rohayem J., Bolognesi M, & Hwu J.R. (2014). Structural Bases of Norovirus RNA Dependent RNA Polymerase Inhibition by Novel Suramin-Related Compounds. PLoS ONE, 9(3), e91765.

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Thermal Stability Assay of Purified Proteins

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Five micrograms of purified protein was mixed with 5× SYPRO orange (Thermo Fisher Scientific) in 20 mM tris-HCl (pH 8.0), 150 mM NaCl at a final volume of 25 μl. The sample mixture was then transferred into an optically clear PCR tube (VWR). SYPRO orange fluorescence data in relative fluorescence unit (RFU) was collected from 10° to 95°C using CFX Connect Real-Time PCR Detection System (Bio-Rad). The temperature corresponding to the lowest point of the first derivative, −d(RFU)/dT, was defined as the melting temperature (T_m). Data were analyzed using OriginPro 2020b (Origin Lab). Raw data are shown in table S5.

Ouyang W.O., Tan T.J., Lei R., Song G., Kieffer C., Andrabi R., Matreyek K.A, & Wu N.C. (2022). Probing the biophysical constraints of SARS-CoV-2 spike N-terminal domain using deep mutational scanning. Science Advances, 8(47), eadd7221.

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Thermal Stability Analysis of MmLipW

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The thermal stability of MmLipW and MmLipW variants was determined using differential scanning fluorimetry.⁴⁵MmLipW protein (0.30 mg/mL) was diluted in triplicate in PBS containing a 1:250 dilution of 5000× Sypro Orange dye (Invitrogen, Carlsbad, CA). The samples were heated from 15 °C to 90 °C at 1.0 °C/min in a thermocycler (Bio-rad C1000 Thermocycler with CFX96 Real-time System, Hercules, CA) and the change in Sypro Orange fluorescence followed over time (λ_ex = 450–490 nm, λ_em = 610–650 nm). The midpoint denaturation temperature (T_m) was determined by plotting the first derivative of fluorescence versus temperature and finding the temperature at the midpoint of the transition.

McKary M.G., Abendroth J., Edwards T.E, & Johnson R.J. (2016). Structural basis for the strict substrate selectivity of the mycobacterial hydrolase LipW. Biochemistry, 55(51), 7099-7111.

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Hsp22 Protein Thermal Stability Assay

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To assess protein thermal stability, Hsp22 WT and Hsp22 NTDΔ were diluted to a final concentration of 5–10 μM in 100 mM sodium acetate buffer pH 7.0 containing 2 mM DTT and 5× Sypro Orange dye (Invitrogen). The 30 μL mixtures were dispensed into a Bio-Rad 96-well thin-wall PCR plate and sealed with microplate adhesive film. Sypro Orange fluorescence was monitored as a function of temperature in a Bio-Rad (Hercules, CA, USA) CFX96 Touch Real-Time PCR Detection System through use of the C1000 Touch Thermal Cycler and FRET channel. Thermal melts were conducted in triplicate for each condition by heating from 25 to 95 °C in 1 °C increments for 2 min and measuring fluorescence at each temperature step. Fluorescence data were blank-subtracted, normalized to the maximum signal for each individual melt, and Boltzmann Sigmoid analysis was conducted using GraphPad Prism to determine the melting temperature (Tm) from two independent experiments.

Webster J.M., Darling A.L., Sanders T.A., Blazier D.M., Vidal-Aguiar Y., Beaulieu-Abdelahad D., Plemmons D.G., Hill S.E., Uversky V.N., Bickford P.C., Dickey C.A, & Blair L.J. (2020). Hsp22 with an N-Terminal Domain Truncation Mediates a Reduction in Tau Protein Levels. International Journal of Molecular Sciences, 21(15), 5442.

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