Circular Dichroism (CD)

• High Quality Quartz cuvette
  • 1 mm pathlength cuvette
    • Hellma 110-1-40, style 110-QS (available from Sigma-Aldrich)
    • Starna 21-Q-1
  • 1 cm pathlength cuvette
    • Hellma 100-10-40, style 100-QS (available from Sigma-Aldrich)
    • Starna 1-Q-10 or 21-Q-10
• your sample
• buffer blank

For Far-UV measurements (protein secondary structure): 1 mm
For Near-UV measurements:  1 cm or 1 mm, depending on application and concentration

Assay Buffers

Buffer selection is critical for accurate CD measurements. Solvent absorbance can severely interfere with the CD signal. Many commonly used buffers 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 no buffer absorbance through the range of the CD spectrum. 
• Always take a scan of your buffer blank to determine whether absorbance interferes with your region of analysis. 
  • Ensure that the buffer blank is well matched to the final dilution of your protein (even trace amounts of some solvents will interfere with the CD measurements).
• Phosphate buffers with little-to-no NaCl are recommended. 10 mM phosphate buffer is ideal. 
• Ideal aqueous solutions contain as little chloride as possible.
  • NaCl and Tris buffers are not recommended but can be tolerated at low concentrations.  
  • If salt is required, SO₄^2- or F^- are preferred counter ions, as they are transparent in the far UV. Potassium fluoride may be a good substitute for chloride-containing salts.
  • Chloride ions will result in a loss of signal below about 200±5 nm, which affects secondary structure estimation, but generally will not block detection of the alpha-helical peaks (208 nm and 222 nm) or the beta-sheet peak (218 nm).
• DTT, ß-ME, or EDTA can be present at low concentrations (1 mM).
• Some detergents are fairly transparent in the far UV (e.g. Chaps and octylglucoside). Triton detergents should be avoided, as they can oxidize readily and form UV-absorbing materials.
• DMSO, formamides, and imidazole absorb strongly in the far UV region, and thus should be avoided in a CD experiment.

Samples

• An accurate protein concentration is required for all CD experiments. 
  • Over-estimating or under-estimating the protein concentration interferes with signal
  • Algorithms that estimate secondary structure depend on correct protein concentrations 
• The amount of sample required for a CD experiment depends on the size, cuvette pathlength and type of measurement being performed.
• 1 mm pathlength cuvettes holds 300 µl and a 1 cm cuvette holds 3 ml.
• Required protein concentration is inversely proportional to the cuvette pathlength, and thus a 1 mm path cuvette requires 10x the concentration of a 1 cm cuvette.
• Recommended concentrations for Far-UV measurements of protein secondary structure are (Jasco J-1500):
  • 0.1 mg/mL in 1 mm path cuvette
  • 0.01 mg/mL in 10 mm patch cuvette
  • Mol/L=115/(MW*7000)*10/pathlength(mm).
  • If possible, make a concentrated stock solution of your protein (at least 2X) and dilute as needed. Protein requirements vary with secondary structure, as alpha-helical proteins have a stronger signal than beta-sheet proteins.
• Protein aggregates can interfere with the CD signal.
  • Assess protein heterogeneity via light scattering.
  • Purify protein samples with soluble aggregates by size-exclusion chromatography.
• Samples can be recovered from the cell. This is not recommended for thermal melts, unless you know your thermal denaturation is reversible.

The absorption and CD spectra should be checked for each buffer.  

Low Wavelength Cutoff For Common Solvents and Buffers in 1mm cuvette

Martin, S. R. Proteins Labfax (Protein, N.C., ed.), Bios Scientific Publishers, Oxford, 1996.

Solvent components not listed here may be compatible but should be tested.  EGTA, DTT and some detergents at modest concentrations may be tolerated.

DichroWeb, a free online tool for determining protein secondary structures based on CD spectra.

• Academic users may apply for a username and password. 

CDToolX, a free, downloadable software program that enables processing and analyses of circular dichroism (CD) spectroscopic data. 

Protein Circular Dichroism Data Bank, a database of CD data

BeStSel (Beta Structure Selection), a free online tool based on a novel method for the secondary structure determination, especially beta-sheet rich proteins.

ValiDichro tests the quality of CD data based on based on common characteristics of CD protein spectra observed in the literature.