The thermostability of TmDAPDC mutants was analyzed using the ThermoFluor assay (Ericsson et al., 2006 (link)) in which the temperature-driven unfolding of a protein is detected by following the increase of fluorescence of Sypro Orange (Molecular Probes, Life Technologies). An enzyme solution (1 mg/ml) was mixed with 100× diluted Sypro Orange dye and heated from 20 to 99°C with a heating rate of 1.75°C/min in a CFX 96 Real Time PCR system (Bio-Rad). Fluorescence was followed with an excitation wavelength of 490 nm and an emission wavelength of 575 nm. Apparent melting temperatures were determined from the derivative of the fluorescence signal vs. temperature.
Sypro orange dye
Manufactured by Bio-Rad
Sourced in United Kingdom, United States
Sypro Orange dye is a fluorescent stain used for the detection and quantification of proteins in polyacrylamide gels. It binds non-covalently to proteins and exhibits an increase in fluorescence upon binding, allowing for sensitive visualization of protein bands.
Automatically generated - may contain errors
Lab products found in correlation
Sypro orange, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time pcr system, by Bio-Rad (1 mentions)
Real time pcr system, by Bio-Rad (1 mentions)
Slide a lyzer mini device, by Thermo Fisher Scientific (1 mentions)
96 well thin wall pcr plate, by Bio-Rad (1 mentions)
Sypro orange dye, by Thermo Fisher Scientific (1 mentions)
Optical quality sealing tape, by Bio-Rad (1 mentions)
Icycler iq real time pcr detection system, by Bio-Rad (1 mentions)
Cfx96 rt pcr system, by Bio-Rad (1 mentions)
Sypro orange dye, by Merck Group (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
6 protocols using sypro orange dye
1
Thermostability Analysis of TmDAPDC Mutants
2
Thermal Stability Assay for FGFR1c
3
Thermal Stability Analysis of COVA2-15 IgA Variants
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The thermal stabilities of the four COVA2-15 IgA1 samples and the IgA2 variant were analysed by a thermal shift assay using a CFX Real-Time PCR system for detection (Bio-Rad, UK). The temperature was held for 10 s plus plate read per degree using 0.1°C increments ranging from 21°C to 95°C. Each sample was prepared and measured in triplicate, containing 10 µg of purified protein and 5 x SYPRO Orange Dye (Bio-Rad, UK) in either 25 µL H2O or purified protein dialyzed in a Slide-A-Lyzer MINI device (Thermo Fisher Scientific, UK) overnight at 4°C in 25 mM Na2HPO4/NaH2PO4 buffer, pH 7.4 and transferred into a standard 96-well PCR plate (Starlabs Semi-Skirted FAST, UK). The resulting curves were fitted using a Boltzmann Equation to identify the melting temperature (Tm) (Huynh and Partch, 2015 (link)).
4
Thermal Stability Assay of Sec3 Protein
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The assay was carried out based on a previously published protocol70 (link). 5,000 × stock solution of Sypro Orange dye (Molecular Probes) was diluted by 300-fold in water. Solutions of 12 μl of protein buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl), 8 μl of Sec3 or Sec3-mutS1 (2.5 mg ml−1) and different amounts of Sypro Orange dye (1, 2, 3, 4 or 5 μl adjusted with water to 5 μl in total) were added onto a 96-well thin-wall PCR plate (Bio-Rad). The plate was then sealed with optical-quality sealing tape (Bio-Rad) and heated in an iCycler iQ real-time PCR detection system (Bio-Rad) from 15 to 95 °C with increments of 0.5 °C per 10 s.
5
Fluorescence Thermal Shift Assay
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Fluorescence thermal shift assay was performed using 10 μg of purified protein and SYPRO orange dye (Bio-Rad, Hercules, CA, USA) using the fluorescence resonance energy transfer channel of the CFX96 RT-PCR System (Bio-Rad, Hercules, CA, USA).
6
Thermostability Assay for IgG Antibodies
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Example 10
The thermostability of XB1511/D8 and XB2202/A4 IgG1 antibodies were determined using a fluorescence based assay. Specifically, 5 mg/ml of purified XB1511/D8 IgG1, XB2202/A4 IgG1, or human IgG1 control were mixed with Sypro orange dye (Sigma) and the temperature of the mixture increased in 1 degree increments from 25° C. to 95° C. The Sypro orange dye incorporates into the IgG when the temperature increases and the IgG unfolds. The fluorescent signal produced by the association of Sypro orange dye with the IgGs was monitored using a BioRad CFX96 instrument. In this assay, the negative regression of the Sypro orange signal was used to identify the peak melting (i.e. Tm) point for each protein.
From this analysis it was determined that XB1511/D8 and XB2202/A4 have melting temperatures (Tm) of 67° C. to 70° C., respectively. This compared well to the human IgG1 control antibody which exhibited a Tm of 72° C. This data demonstrate that the VH and VH/VL pairs of the invention are capable of being formatted into highly thermostable full-length IgG molecules.
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