Several instrument parameters can be adjusted to improve signal to noise by increasing the amount of light measured at a given
wavelength (data pitch, scan mode, scan speed, and response time)
(see Note 9). A quick, preliminary scan is helpful in guiding parameter adjustment for new proteins. Usually, the CD signal, i.e., the
ellipticity θ, the high tension (HT) voltage (or gain), and the
absorbance are recorded. The detector will have a limited range of
amplification in which it is linear; thus, the HT voltage, which
controls the gain of the detector on most laboratory instruments,
should remain below 600 V (or manufacturer-specified limit). If it
is too high, you will need to reduce absorbance by decreasing buffer
and/or protein concentration or path length.
Measurement of a CD
Spectrum
A CD spectrum recorded in the far-UV region will produce a
spectrum that has a characteristic shape and magnitude determined
by the secondary structure elements of the protein. Even a
low-quality spectrum can readily distinguish folded from unfolded
protein. However, any spectral comparisons used to monitor structural changes (e.g., due to solution conditions or batch-to-batch
variation or calculation of secondary structure content) will require
care to optimize spectral quality. A baseline spectrum must be
collected of the buffer (the actual buffer used to prepare the protein) and the cuvette inserted into the instrument in the same
orientation as for the sample measurement. The baseline spectrum
must be recorded with the same instrument parameters as the
subsequent protein sample. Usually, a 1-mm cuvette is used, requiring ~200 μL of buffer and protein solution (mininum 160 μl,
maximum 350 μl). As experience is built, it is a good idea to create
a list of usable path lengths and instrument settings for particular
buffers and proteins. A comparative analysis of spectra is facilitated
by reusing the same experimental parameters. All comparison
should be carried out in terms of molar ellipticity and thus will
require correction for any differences in protein concentration (see
Note 10).
A simple protocol to record a CD spectrum is given below:
1. Purge the instrument with N 2 according to the manufacturer’s
instructions (usually 10–15 min).
2. Switch on the instrument (and the temperature control/water
bath, if necessary).
3. Switch on the lamp and let it warm up for 10–20 min.
4. Set appropriate instrument parameters following the manufacturer’s instruction (see Note 9).
5. Record a spectrum of the empty 1-mm quartz cuvette (a single
accumulation is sufficient). Even slight characteristics of a protein spectrum for the empty cuvette indicate contamination
from previous experiments, and thorough cleaning of the
32
Bertrand Raynal et al.
wavelength (data pitch, scan mode, scan speed, and response time)
(see Note 9). A quick, preliminary scan is helpful in guiding parameter adjustment for new proteins. Usually, the CD signal, i.e., the
ellipticity θ, the high tension (HT) voltage (or gain), and the
absorbance are recorded. The detector will have a limited range of
amplification in which it is linear; thus, the HT voltage, which
controls the gain of the detector on most laboratory instruments,
should remain below 600 V (or manufacturer-specified limit). If it
is too high, you will need to reduce absorbance by decreasing buffer
and/or protein concentration or path length.
Measurement of a CD
Spectrum
A CD spectrum recorded in the far-UV region will produce a
spectrum that has a characteristic shape and magnitude determined
by the secondary structure elements of the protein. Even a
low-quality spectrum can readily distinguish folded from unfolded
protein. However, any spectral comparisons used to monitor structural changes (e.g., due to solution conditions or batch-to-batch
variation or calculation of secondary structure content) will require
care to optimize spectral quality. A baseline spectrum must be
collected of the buffer (the actual buffer used to prepare the protein) and the cuvette inserted into the instrument in the same
orientation as for the sample measurement. The baseline spectrum
must be recorded with the same instrument parameters as the
subsequent protein sample. Usually, a 1-mm cuvette is used, requiring ~200 μL of buffer and protein solution (mininum 160 μl,
maximum 350 μl). As experience is built, it is a good idea to create
a list of usable path lengths and instrument settings for particular
buffers and proteins. A comparative analysis of spectra is facilitated
by reusing the same experimental parameters. All comparison
should be carried out in terms of molar ellipticity and thus will
require correction for any differences in protein concentration (see
Note 10).
A simple protocol to record a CD spectrum is given below:
1. Purge the instrument with N 2 according to the manufacturer’s
instructions (usually 10–15 min).
2. Switch on the instrument (and the temperature control/water
bath, if necessary).
3. Switch on the lamp and let it warm up for 10–20 min.
4. Set appropriate instrument parameters following the manufacturer’s instruction (see Note 9).
5. Record a spectrum of the empty 1-mm quartz cuvette (a single
accumulation is sufficient). Even slight characteristics of a protein spectrum for the empty cuvette indicate contamination
from previous experiments, and thorough cleaning of the
32
Bertrand Raynal et al.