Procedure:

Purge the CD spectropolarimeter optics compartment with ultrapure nitrogen gas at ~10 litres/minute for 15 minutes (As long as the instrument is on, there should be uninterrupted N₂ supply).
Turn ON the lamp of the spectropolarimeter.
Turn ON the other parts of the spectropolarimeter and the computer; allow 30 minutes warm up.
Open the spectra collecting software.
Set the half bandwidth between 1 – 1.5 nm.
Set the wavelength range: wavelength range from 260 – 185 nm is suitable for the 0.1 – 0.2 mg/ml protein solutions in the buffers that don't absorb in this range.
Set the number of scans to 8 (This means that the final CD spectrum will be an average of 8 different scans).
Define the path in the software for storing the data.
Set the wavelength interval: for samples with signal to noise ratio > 20:1, 0.5 nm is an optimum interval; if the signal to noise ratio is low, the interval can be set at 0.1 or 0.2 nm.
Set the data collection time at each wavelength to 1 second .
Set the instrument time constant to 100 ms (Note 3).
Set the instrument to record the ellipticity and the PMT voltage (Note 4).
Take 200 μl of filtered phosphate buffer in the 1 mm path length quartz cuvette.
Record the CD while monitoring the PMT voltage (PMT voltage increases as the instrument scans lower wavelengths) (Note 4). If the PMT goes above 500 V, the buffer may not be suitable for the lower wavelengths.
Remove the buffer from the cuvette and add 200 μl of 20 μg/ml lysozyme solution.
If PMT voltage goes above 500 V, the spectrum becomes noisy and less reliable. In that case, the protein solution needs to be diluted or the spectra be recorded to a higher wavelength, say 190 or 195 nm.
Remove the buffer from the cuvette and add 200 μl of 20 μg/ml lysozyme solution.
Record the CD spectrum for the protein solution.
Subtract the blank spectrum from the recorded protein spectrum to obtain the corrected protein spectrum.
Save the corrected spectrum as a separate text file.