CD: Circular Dichroism

CD measures the difference in absorption between left and right handed circularly polarized light of chiral samples. CD is an established biophysical method used to study folding of peptides, proteins, oligo-nucleotides and DNA which have distinct CD bands in the far-UV and VUV. Secondary structure can be determined and changes followed with e.g. ligand binding, temperature, salinity etc.

What are the sample requirements?

  • The protein concentration required is inversely proportional to the cuvette pathlength, and thus a 1 mm path cuvette requires 10x the concentration of a 10 mm cuvette.
  • Typical concentrations of samples required are: 1 mg/ml for 0.1 mm pathlength, 1 mg/ml for 1 mm pathlength, 10 mg/ml for 10 mm pathlength
    with alpha-helical proteins possibly needing ~half this concentration, while predominantly beta-sheet may require double this concentration. If possible, make a concentrated stock solution of your protein (at least 2x) and dilute as needed.
  • Load volumes are typically 30-150 uL for 0.1 mm pathlength, 350-500 uL for 1 mm and 3 mL for a 10 mm pathlength cell (these depend on the type of cell available and whether a temperature melt is being measured).
  • Many commonly used buffers (Tris, HEPES, MOPS) and additives absorb in the far UV region used for CD measurements. The ideal CD experiment is performed in a buffer in which your protein is well behaved and soluble with little to no buffer absorbance through the range of the CD spectrum. The optimum buffer is a phosphate buffer, pH adjusted using a mixture of the mono and divalent Na+ forms, with ionic strength adjusted using NaF instead of NaCl.

What other specific considerations are relevant?

  • Protein aggregates can interfere with the CD signal. It may be necessary to assess protein heterogeneity via light scattering and purify protein samples with soluble aggregates by size-exclusion chromatography.
  • Measurement of protein aggregates or highly scattering samples may be possible, for samples of this kind you can contact AU-SRCD.
  • For determination of secondary structure, an accurate measurement of concentration of the protein/peptide is needed.

Partners offering this technique

MOSBRI reference partner site for this technique:

Other partners: