DSF measurements
were performed using a StepOnePlus real-time quantitative PCR instrument
(Applied Biosystems) and software (version 2.3). DSF assays (20 μL)
were run in duplicate in sealed MicroAmp Fast 96-well qPCR plates
(Applied Biosystems). DSF profiles were acquired with recombinant
Abl core proteins (2 μM) in bicine buffer (10 mM bicine, 150
mM NaCl, pH 8.0) and SYPRO Orange (Sigma) diluted to a 5× working
concentration. Parallel reactions without proteins were run to correct
for background fluorescence. DSF reactions were allowed to equilibrate
to 25 °C for 2 min, followed by an increase to 99 °C at
a 1% temperature ramp rate (1.6 °C/min) with continuous data
collection. Background fluorescence was subtracted, and mean fluorescence
intensities were then plotted as a function of temperature. Melt curves
were fit using the Boltzmann sigmoid function of GraphPad Prism 6,
and Tm values were calculated as the midpoint
of the thermal transition between the minimum and maximum fluorescence
intensities.
were performed using a StepOnePlus real-time quantitative PCR instrument
(Applied Biosystems) and software (version 2.3). DSF assays (20 μL)
were run in duplicate in sealed MicroAmp Fast 96-well qPCR plates
(Applied Biosystems). DSF profiles were acquired with recombinant
Abl core proteins (2 μM) in bicine buffer (10 mM bicine, 150
mM NaCl, pH 8.0) and SYPRO Orange (Sigma) diluted to a 5× working
concentration. Parallel reactions without proteins were run to correct
for background fluorescence. DSF reactions were allowed to equilibrate
to 25 °C for 2 min, followed by an increase to 99 °C at
a 1% temperature ramp rate (1.6 °C/min) with continuous data
collection. Background fluorescence was subtracted, and mean fluorescence
intensities were then plotted as a function of temperature. Melt curves
were fit using the Boltzmann sigmoid function of GraphPad Prism 6,
and Tm values were calculated as the midpoint
of the thermal transition between the minimum and maximum fluorescence
intensities.