starting point is to employ 100–150μg/mL of protein in a
low-absorbance buffer. With this path length, lower concentrations
can be used successfully by collecting data for longer, but very high
concentrations (>300μg/mL) will absorb too much light. For
near-UV experiments where buffer and protein absorbance (dependent on aromatic content) is smaller and the CD signal is also
smaller, higher concentrations and/or longer path lengths (e.g.,
1 cm) will typically be used.
A low concentration phosphate buffer with little salt (e.g.,
5 mM phosphate, 5 mM NaF) is recommended as a good low-absorbance spectroscopic buffer. However, in protein QC, it is
most important to work with solution conditions in which the
protein is well behaved, and which will be used for downstream
applications. These will not necessarily be optimal from a spectroscopy perspective. To carry out CD experiments, the buffer alone
must not have UV absorbance >1 (and preferably much lower)
anywhere in the range of the spectrum, as it will, otherwise, absorb
most of the light. Consequently, buffers with compounds containing aromatic groups cannot be employed. High concentrations of
chloride ions absorb at wavelengths <215 nm and should thus be
avoided. If high salt is required, chloride may be replaced by
fluoride. Many components of the sample buffer may prevent
reliable recording near or below 200 nm (see Note 7), compromising the reliability of secondary structure calculation; the validity of
spectral comparison, however, is not affected. It is possible to
reduce the detrimental effects of absorbing buffers on measurements by working with higher protein concentration in shorter
path-length cuvettes.
Scattering from contaminants/aggregates is indicated by a
nonflat baseline at wavelengths longer than those where the sample
absorbs light (for far-UV >250 nm, for near-UV >320 nm). The
buffer should be filtered (0.1 or 0.02μm), and the sample should be
centrifuged (10 min at full speed in a benchtop centrifuge); both
the buffer and the sample should be degassed. Further details on
the choice of cuvettes and buffers as well as sample preparation can
be found in Ref. 18.
Spectrometer Setup
CD spectrometers must be purged continuously with nitrogen
(1) to exclude O 2 from the sample compartment as O 2 absorbs
incident radiation, which, in turn, limits the lowest wavelength that
can be measured and (2) to prevent the production of ozone by the
lamp, which would damage the optics. To ensure robust quantitative analysis or comparison of samples over time, the spectrometer
should be calibrated on a weekly basis to validate wavelength accuracy, wavelength repeatability, intensity accuracy, intensity repeatability, baseline flatness, baseline stability, and the noise level (for
details and protocols, see instrument manuals and Ref. 18).
Protein Quality Control
31
low-absorbance buffer. With this path length, lower concentrations
can be used successfully by collecting data for longer, but very high
concentrations (>300μg/mL) will absorb too much light. For
near-UV experiments where buffer and protein absorbance (dependent on aromatic content) is smaller and the CD signal is also
smaller, higher concentrations and/or longer path lengths (e.g.,
1 cm) will typically be used.
A low concentration phosphate buffer with little salt (e.g.,
5 mM phosphate, 5 mM NaF) is recommended as a good low-absorbance spectroscopic buffer. However, in protein QC, it is
most important to work with solution conditions in which the
protein is well behaved, and which will be used for downstream
applications. These will not necessarily be optimal from a spectroscopy perspective. To carry out CD experiments, the buffer alone
must not have UV absorbance >1 (and preferably much lower)
anywhere in the range of the spectrum, as it will, otherwise, absorb
most of the light. Consequently, buffers with compounds containing aromatic groups cannot be employed. High concentrations of
chloride ions absorb at wavelengths <215 nm and should thus be
avoided. If high salt is required, chloride may be replaced by
fluoride. Many components of the sample buffer may prevent
reliable recording near or below 200 nm (see Note 7), compromising the reliability of secondary structure calculation; the validity of
spectral comparison, however, is not affected. It is possible to
reduce the detrimental effects of absorbing buffers on measurements by working with higher protein concentration in shorter
path-length cuvettes.
Scattering from contaminants/aggregates is indicated by a
nonflat baseline at wavelengths longer than those where the sample
absorbs light (for far-UV >250 nm, for near-UV >320 nm). The
buffer should be filtered (0.1 or 0.02μm), and the sample should be
centrifuged (10 min at full speed in a benchtop centrifuge); both
the buffer and the sample should be degassed. Further details on
the choice of cuvettes and buffers as well as sample preparation can
be found in Ref. 18.
Spectrometer Setup
CD spectrometers must be purged continuously with nitrogen
(1) to exclude O 2 from the sample compartment as O 2 absorbs
incident radiation, which, in turn, limits the lowest wavelength that
can be measured and (2) to prevent the production of ozone by the
lamp, which would damage the optics. To ensure robust quantitative analysis or comparison of samples over time, the spectrometer
should be calibrated on a weekly basis to validate wavelength accuracy, wavelength repeatability, intensity accuracy, intensity repeatability, baseline flatness, baseline stability, and the noise level (for
details and protocols, see instrument manuals and Ref. 18).
Protein Quality Control
31