Experimental Guidelines

Experimental Design

To measure CD spectra, 300 microliters of protein solution with concentration no less than 0.2 mg/ml and several ml of buffer used for this protein has to be sent to the Director of the facility.

Protein solution should contain only those chemicals necessary to maintain protein stability, and at the lowest concentrations possible.Avoid any chemical that is unnecessary for protein stability/solubility. The protein itself should be as pure as possible, any additional protein or peptide will contribute to the CD signal. Filtering of the solutions (0.02 um syringe filters) may improve the signal-to-noise ratio.

Any compound which absorbs in the region of interest (190-260 nm) should be avoided. A buffer or detergent or other chemical should not be used unless it can be shown that the compound in question will not mask the protein signal. Ensure that only the minimum concentrations of additives are present in the protein solution.

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Buffer Properties

• Buffer Lower wavelength limit, nm*
• 10 mM potassium phosphate, 100 mM potassium fluoride 185
• 10 mM potassium phosphate, 100 mM (NH4)2SO4 185
• 10 mM potassium phosphate, 50 mM Na2SO4 185
• 10 mM potassium phosphate, 100 mM KCl 195
• 20 mM sodium phosphate, 100 mM NaCl 195
• Dulbecco’s phosphate buffered saline (PBS): 200
• (9.33 mM K-phosphate, 136 mM NaCl, 2.7 mM KCl, 0.6 mM MgCl2, 0.9 mM CaCl2)
• 2 mM Hepes, 50 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol 200
• 50 mM Tris, 150 mM NaCl, 1 mM dithiothreitol, 0.1 mM EDTA 201

*The lower limit values are typical for solutions containing B 0.1 mg ml–1 protein in 0.1-cm cells. Below the lower wavelength cutoffs the dynode voltages rapidly increase, the signal to noise is poor and the ellipticity is not a linear function of the path length of the cell. DMSO and formamides have high absorbance and cannot be used for CD measurements. Many organic solvents, e.g. trifluoroethanol, hexafluoropropanol and hexane are transparent to 185 nm and below but will change the structure of proteins and polypeptides. Buffers can contain up to 20% glycerol, but measurements can only be made to 200 nm at this concentration.