DSF experiments were performed on an Applied Biosystems ViiA 7 qPCR machine with excitation and emission wavelengths set to 470 nm and 569 nm with proteins in buffer of 100 mM NaCl, 20 mM sodium phosphate pH 7.2. Experiments were conducted in triplicate in MicroAmp Fast 96-well plates with 50 μL total volume containing final concentrations of 7 μM protein and 10× SYPRO orange dye (ThermoFisher). For UV-treated samples, proteins were UV-irradiated at 365 nm for 1 hour at room temperature and assayed following centrifugation at 13,000 rpm for 10 minutes to remove precipitates. For reducing agent treated samples, samples were incubated with 1 mM TCEP for 1 hour and assayed in the presence of TCEP, following centrifugation at 13,000 rpm for 10 minutes to remove precipitates. Rescue experiments were performed by incubation with 20-fold molar excess peptide throughout the duration of the experiment. Temperature was incrementally increased at a scan rate of 1°C/min between 25°C and 95°C. Data analysis and fitting was performed in GraphPad Prism v7.
Microamp fast 96 well plates
Manufactured by Thermo Fisher Scientific
The MicroAmp Fast 96-well plates are a laboratory consumable product designed for use in qPCR (quantitative Polymerase Chain Reaction) and other real-time PCR applications. The plates feature a 96-well format and are compatible with standard thermal cyclers.
Automatically generated - may contain errors
6 protocols using microamp fast 96 well plates
1
Differential Scanning Fluorimetry of Proteins
2
Differential Scanning Fluorimetry of Proteins
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DSF experiments were performed on an Applied Biosystems ViiA 7 qPCR machine with excitation and emission wavelengths set to 470 nm and 569 nm with proteins in buffer of 100 mM NaCl, 20 mM sodium phosphate pH 7.2. Experiments were conducted in triplicate in MicroAmp Fast 96-well plates with 50 μL total volume containing final concentrations of 7 μM protein and 10× SYPRO orange dye (ThermoFisher). For UV-treated samples, proteins were UV-irradiated at 365 nm for 1 hour at room temperature and assayed following centrifugation at 13,000 rpm for 10 minutes to remove precipitates. For reducing agent treated samples, samples were incubated with 1 mM TCEP for 1 hour and assayed in the presence of TCEP, following centrifugation at 13,000 rpm for 10 minutes to remove precipitates. Rescue experiments were performed by incubation with 20-fold molar excess peptide throughout the duration of the experiment. Temperature was incrementally increased at a scan rate of 1°C/min between 25°C and 95°C. Data analysis and fitting was performed in GraphPad Prism v7.
3
Thermal Stability Profiling of Proteins
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DSF experiments were performed on an Applied Biosystems ViiA 7 qPCR machine with excitation and emission wavelengths set to 470 nm and 569 nm with proteins in buffer of 50 mM NaCl, 20 mM sodium phosphate pH 7.2. Experiments were conducted in triplicate in MicroAmp Fast 96-well plates with 50 μL total volume containing final concentrations of 7 μM protein and 10× SYPRO orange dye (ThermoFisher). The temperature was incrementally increased at a scan rate of 1 °C/min between 25 °C and 95 °C. Data analysis and fitting were performed in GraphPad Prism v7.
4
Thermal Stability Assay for pMHC-I
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To measure the thermal stability of the pMHc-I molecules, 2.5 μM of protein was mixed with 10× Sypro Orange dye in matched buffer (20 mM sodium phosphate pH 7.2, 100 mM NaCl) in MicroAmp Fast 96-well plates (Applied Biosystems) at a final volume of 50μl. Differential scanning fluorimetry was performed using an Applied Biosystems ViiA quantitative PCR machine with excitation and emission wavelengths at 470 nm and 569 nm, respectively. Thermal stability was measured by increasing the temperature from 25 °C to 95 °C at a scan rate of 1 °C min−1. Melting temperatures (Tm) were calculated in GraphPad Prism 7 by plotting the first derivative of each melt curve and taking the peak as the Tm.
5
Thermal Stability Assay of pMHc-I
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To measure the thermal stability of the pMHc-I molecules, 2.5 μM of protein was mixed with 10× Sypro Orange dye in matched buffer (20 mM sodium phosphate pH 7.2, 100 mM NaCl) in MicroAmp Fast 96-well plates (Applied Biosystems) at a final volume of 50μl. Differential scanning fluorimetry was performed using an Applied Biosystems ViiA quantitative PCR machine with excitation and emission wavelengths at 470 nm and 569 nm, respectively. Thermal stability was measured by increasing the temperature from 25 °C to 95 °C at a scan rate of 1 °C min−1. Melting temperatures (Tm) were calculated in GraphPad Prism 7 by plotting the first derivative of each melt curve and taking the peak as the Tm.
6
Thermal Stability Assay for pMHC-I Complexes
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To measure thermal stability of pMHC-I molecules37 (link), 2.5 μM of protein was mixed with 10 x Sypro Orange dye in matched buffers (20 mM sodium phosphate pH 7.2, 100 mM NaCl) in MicroAmp Fast 96 well plates (Applied Biosystems) at a final volume of 50 μL. DSF was performed using an Applied Biosystems ViiA qPCR machine with excitation and emission wavelengths at 470 nm and 569 nm respectively. Thermal stability was measured by increasing the temperature from 25 °C to 95 °C at a scan rate of 1 °C/min. Melting temperatures (Tm) were calculated in GraphPad Prism 7 by plotting the first derivative of each melt curve and taking the peak as the Tm (Supplementary Fig. 9 a). Determination of Tm values of TAPBPR-exchanged molecules additionally required subtraction of the TAPBPR melt curve from the curve obtained for the complex, then calculating the first derivative. This procedure, on average, enhanced the Tm values calculated for TAPBPR-exchanged pMHC-I molecules by 1.5 °C, compared to refolded and photo-exchanged pMHC-I molecules. All samples were analyzed in duplicate and the error is represented as the standard deviation of the duplicates analyzed independently.
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