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Thermal denaturation by CD spectroscopy

JP Juan Luis Pacheco-García
DL Dmitry S. Loginov
AN Athi N. Naganathan
PV Pavla Vankova
MC Mario Cano-Muñoz
PM Petr Man
AP Angel L. Pey
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For thermal denaturation experiments by CD spectroscopy, we used exactly the same samples used to acquire CD wavelength-dependent spectra. Samples were thermostatized at 20 °C for 10 min and then temperature was raised to 75 °C at a 2 °C min−1 scan rate. Signals were acquired at 222 nm (for UV-CD in 1 mm path-length cuvettes) or 290 nm (for Near-UV CD in 5 mm path-length), using a bandwidth of 2 nm and a time response of 16 s. Raw data were normalized for linear pre- and post-transition baselines and the apparent Tm was determined as the temperature at which half of the unfolded fraction was observed by linear extrapolation. Thermal denaturation leads to irreversible protein aggregation (i.e. formation of protein aggregates of large size)30.

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