cuvette is required, either by rinsing with water and drying with
methanol or, if that is ineffective cleaning, with an appropriately diluted Hellmanex solution according to the manufacturer’s protocol (e.g., 0.5–2% (v/v) Hellmanex for
20–180 min at 25–30
C) or HCl. Remeasure the cuvette
spectrum after cleaning.
6. Filter and degas the buffer. Fill the cuvette with 200 μL of
buffer and record a reference spectrum.
7. Remove liquid and dry the cuvette.
8. Prepare the sample solution, centrifuge the sample in a benchtop centrifuge for 5–10 min at full speed, and degas it.
9. Fill the cuvette with 200 μL of sample solution and record a
spectrum (same parameters as in step 5). Note that the sample
spectrum has to be recorded with the same cuvette as the
reference/buffer spectrum as the cuvette influences the background more strongly than the buffer.
10. Repeat steps 4–10 as required for additional samples or to
optimize instrument parameters, concentrations, etc. (or for
additional samples).
11. Switch off the lamp and the instrument, and purge with N 2
according to the manufacturer’s instruction (usually 15–20
min).
12. Switch off N 2 flux.
Estimation of Secondary
Structure Composition
The CD spectrum in the far-UV region (throughout 180–250 nm)
is largely determined by the secondary structure elements of the
protein. The fraction of each secondary structure element can be
approximated using different methods, which all make a linear
combination of a set of reference spectra, in the simplest case
spectra of purely α-helical, β-sheet, and random-coil structures
(Fig. 6), plus a noise term containing the contribution of aromatic
chromophores and prosthetic groups. Analysis may be carried out
by either instrument-specific software or online (e.g., using the
Beta Structure Selection (BeStSel) server (http://bestsel.elte.hu))
[47, 48]. An overview of different methods can be found in Ref.
18. Bear in mind that absolute accuracy of methods is very dependent on data quality, particularly at low wavelengths (where buffers
often interfere). However, even without absolute accuracy, values
are useful in comparative analysis, e.g., of samples, mutants, and
denaturation.
Determination of Protein
Thermostability
Prior to a thermal denaturation experiment, far-UV CD spectra of
the sample should be recorded at 20
C and any parameters optimized as necessary (as above). Ideally, a thermal denaturation
experiment should be carried out in a magnetically stirred 10-mm
quartz cuvette in order to enable the use of a low protein
Protein Quality Control
33
methanol or, if that is ineffective cleaning, with an appropriately diluted Hellmanex solution according to the manufacturer’s protocol (e.g., 0.5–2% (v/v) Hellmanex for
20–180 min at 25–30
C) or HCl. Remeasure the cuvette
spectrum after cleaning.
6. Filter and degas the buffer. Fill the cuvette with 200 μL of
buffer and record a reference spectrum.
7. Remove liquid and dry the cuvette.
8. Prepare the sample solution, centrifuge the sample in a benchtop centrifuge for 5–10 min at full speed, and degas it.
9. Fill the cuvette with 200 μL of sample solution and record a
spectrum (same parameters as in step 5). Note that the sample
spectrum has to be recorded with the same cuvette as the
reference/buffer spectrum as the cuvette influences the background more strongly than the buffer.
10. Repeat steps 4–10 as required for additional samples or to
optimize instrument parameters, concentrations, etc. (or for
additional samples).
11. Switch off the lamp and the instrument, and purge with N 2
according to the manufacturer’s instruction (usually 15–20
min).
12. Switch off N 2 flux.
Estimation of Secondary
Structure Composition
The CD spectrum in the far-UV region (throughout 180–250 nm)
is largely determined by the secondary structure elements of the
protein. The fraction of each secondary structure element can be
approximated using different methods, which all make a linear
combination of a set of reference spectra, in the simplest case
spectra of purely α-helical, β-sheet, and random-coil structures
(Fig. 6), plus a noise term containing the contribution of aromatic
chromophores and prosthetic groups. Analysis may be carried out
by either instrument-specific software or online (e.g., using the
Beta Structure Selection (BeStSel) server (http://bestsel.elte.hu))
[47, 48]. An overview of different methods can be found in Ref.
18. Bear in mind that absolute accuracy of methods is very dependent on data quality, particularly at low wavelengths (where buffers
often interfere). However, even without absolute accuracy, values
are useful in comparative analysis, e.g., of samples, mutants, and
denaturation.
Determination of Protein
Thermostability
Prior to a thermal denaturation experiment, far-UV CD spectra of
the sample should be recorded at 20
C and any parameters optimized as necessary (as above). Ideally, a thermal denaturation
experiment should be carried out in a magnetically stirred 10-mm
quartz cuvette in order to enable the use of a low protein
Protein Quality Control
33