| | Re: Protein Folding
There can be several reasons. Did you do the experiments under exactly the same conditions? That is, was the buffer composition the same? Was the protein you used from the same purification batch?
Another reason for the differences could be due to your CD measurement. Did you measure far-UV or near-UV CD spectra, or both? Far-UV gives you information about secondary structure. It has long been known that for most proteins, secondary structure elements persist even when the protein has begun to unfold. This is called the 'molten globule' state. Near-UV CD gives more information about the tertiary structure, and should correlate better with DSC measurements, which would (if my understanding is correct) give a peak when the molten globule forms.
But an unfortunate fact about biological molecules is that different types of measurement can give different results... I agree that in theory thermal unfolding should give the same Tm regardless of measurement, but this is sadly not always the case... Other methods you could think about for measuring the Tm are monitoring intrinsic fluorescence or using fluorescent dyes (e.g. in Thermofluor) to get an estimate of the Tm. These should be closer to the value that DSC gives, as they are based on changes in tertiary structure and solvent exposure, rather than secondary structure as in far-UV CD.
Last edited by JLeo; 06-29-2010 at 10:30 AM.