Consequently, samples should not be concentrated beyond the
absolutely required concentrations, avoiding overly high concentrations. Moreover, concentration of the samples should be
followed by analytical SEC or DLS using an aliquot of the
concentrated sample to ensure that it does not contain
aggregates.
5. When measurements are performed with short path lengths,
small manufacturing errors in path length will represent a larger
percentage of the measurement; consequently, for quantitative
analysis, short path-length cuvettes should be calibrated.
Equipment that is cuvette-free (such as a Nanodrop (Thermo
Scientific)) should be checked and maintained regularly for
path length accuracy. Furthermore, be aware that the path
length of such instruments may be disturbed by highly concentrated protein or glycerol-containing samples because of
their high viscosity.
6. Buffer mismatch can be detected when the absorbance signal
remains constant between 320 and 340 nm but not equal to
zero. This is due to the buffer difference between the blank and
the sample and should be a difference of not more than a few
mAU. In that case, the baseline absorbance can be adjusted to
zero, using the absorbance at 340 nm. See the manufacturer’s
instructions for the procedure. However, be aware that some
software may use this option as default setting, which should be
avoided! Similarly, if scattering affects the overall absorbance,
the contribution of scattering can be removed by tracing a log–
log plot of absorbance versus wavelength in the 320–340 nm
region and then extrapolating the curve to the rest of the
spectrum [1, 42]. The resulting value at 280 nm can then be
removed from the A 280 signal in order to calculate the concentration. However, concentration measurements will be less
accurate if this correction is applied, and it should be avoided
for measurements at 205 or 214 nm because the greater extrapolation will be more inaccurate.
7. Of commonly used buffers, phosphate, borate, MES, MOPS,
and PIPES have relatively low absorbance in the far-UV, TRIS
and HEPES have moderate absorbance and can only be used in
low concentration, and DTT, 2-mercaptoethanol, and DMSO
have very high absorbance in the main region of the far-UV CD
signal (200–230 nm) and cannot be used in CD samples. If a
reducing agent is required, TCEP has moderate absorbance.
See http://www.uslims.aucsolutions.com/labresources.php
for absorption spectra.
8. For other staining approaches to detect smaller quantities of
the target protein or contaminants, we recommend the use of
commercial staining kits as their protocols and formulations are
Protein Quality Control
41
absolutely required concentrations, avoiding overly high concentrations. Moreover, concentration of the samples should be
followed by analytical SEC or DLS using an aliquot of the
concentrated sample to ensure that it does not contain
aggregates.
5. When measurements are performed with short path lengths,
small manufacturing errors in path length will represent a larger
percentage of the measurement; consequently, for quantitative
analysis, short path-length cuvettes should be calibrated.
Equipment that is cuvette-free (such as a Nanodrop (Thermo
Scientific)) should be checked and maintained regularly for
path length accuracy. Furthermore, be aware that the path
length of such instruments may be disturbed by highly concentrated protein or glycerol-containing samples because of
their high viscosity.
6. Buffer mismatch can be detected when the absorbance signal
remains constant between 320 and 340 nm but not equal to
zero. This is due to the buffer difference between the blank and
the sample and should be a difference of not more than a few
mAU. In that case, the baseline absorbance can be adjusted to
zero, using the absorbance at 340 nm. See the manufacturer’s
instructions for the procedure. However, be aware that some
software may use this option as default setting, which should be
avoided! Similarly, if scattering affects the overall absorbance,
the contribution of scattering can be removed by tracing a log–
log plot of absorbance versus wavelength in the 320–340 nm
region and then extrapolating the curve to the rest of the
spectrum [1, 42]. The resulting value at 280 nm can then be
removed from the A 280 signal in order to calculate the concentration. However, concentration measurements will be less
accurate if this correction is applied, and it should be avoided
for measurements at 205 or 214 nm because the greater extrapolation will be more inaccurate.
7. Of commonly used buffers, phosphate, borate, MES, MOPS,
and PIPES have relatively low absorbance in the far-UV, TRIS
and HEPES have moderate absorbance and can only be used in
low concentration, and DTT, 2-mercaptoethanol, and DMSO
have very high absorbance in the main region of the far-UV CD
signal (200–230 nm) and cannot be used in CD samples. If a
reducing agent is required, TCEP has moderate absorbance.
See http://www.uslims.aucsolutions.com/labresources.php
for absorption spectra.
8. For other staining approaches to detect smaller quantities of
the target protein or contaminants, we recommend the use of
commercial staining kits as their protocols and formulations are
Protein Quality Control
41