Circular Dichroism (CD)

Circular Dichroism (CD) Spectroscopy is used to determine the optical isomerism and secondary structure of molecules.  Circular dichrosim (measured in molar ellipticity) is the difference in absorption of left-handed and right-handed circularly polarized light and can be observed in molecules with chiral centers.  CD spectra in the "far UV" region (185-250 nm) can be used to determine protein secondary structure.  Thermal stability (Tm) can be measured by following changes in molar ellipticity with increasing temperature.

 

The CMI has a Jasco J-815 CD Spectropolarimeter with a Peltier temperature controller and single cuvette holder.

For information on access fees, policies and getting started at the CMI, see the CMI Access Page.

CMI Getting Started with CD for aid in preparing for your first experiment
CMI Jasco J-815 Standard Protocol for a basic protocol for far-UV CD.

Protein Circular Dichroism Data Bank, a database of CD data

Dichroweb, an online circular dichroism analysis suite

  

The J-815 User Guide is available at the instrument, but not electronically.

  • High Quality Quartz cuvette
    • Far-UV measurements (protein secondary structure):  1 mm pathlength cuvette (eg. Hellma 110-1-40, style 110-QS, available from Sigma-Aldrich)
    • Near-UV measurements:  1 cm pathlength cuvette (eg. Hellma 100-10-40, style 100-QS, available from Sigma-Aldrich)
  • your sample
  • buffer blank

 

Protein Sample

Recommended protein concentration depends on the size of your protein, the pathlength of the cuvette, and the type of measurement.  For standard "Far-UV" measurements of protein secondary structure, the following protein concentrations are a good place to start:  

  • 0.2 mg/ml in 1 mm path cuvette
  • 0.02 mg/ml in 10 mm patch cuvette                    

OR

  • Mol/L=115/(MW*7000)*10/pathlength(mm)

 

  • concentration is inversely proportional to the cuvette pathlength, so a 1 mm path cuvette requires 10x the concentration of a 1 cm cuvette.
  • A standard 1 mm pathlength cuvette holds 300ul and a 1 cm cuvette holds 3 ml.  
  • For alpha-helical proteins you may need ½ this concentration, for beta-sheet proteins, you may need double the concentration.  If possible, make a concentrated stock solution (at least 2X) and dilute as needed.
  • Sample can be recovered from the cell (not recommended for thermal melts unless you know your thermal denaturation is reversible).

 

Buffer

Buffer selection is important for accurate CD measurements as solvent absorbance can interfere with the CD signal.  Ideally you will have no solvent absorbance throughout the range of the CD spectrum, typically 185-260nm for protein samples.  Many commonly used buffers and additives absorb in the far UV region used for CD measurements.    It's always important to keep your protein well behaved and soluble but finding a solvent that is compatible with CD is also required.

Always take a scan of your buffer alone (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).

  • Phosphate buffers with little to no NaCl are recommended.  10mM phosphate is an excellent choice.  If salt is required, SO42- or F- are preferred counter ions, as they are transparent in the far UV.
  • DTT, BME, or EDTA can be present at low concentrations (1mM).
  • Some detergents are fairly transparent in the far UV (eg. Chaps and octylglucoside). Triton detergents should be avoided, as they can oxidize readily and form UV-absorbing materials.
  • Ideal aqueous solutions contain as little chloride as possible so NaCl and Tris buffers are not recommended, but can be tolerated at low concentrations.  
    • If a high ionic strength is needed, Potassium fluoride may be a good substitute for chloride containing salts.
    • The presence of chloride ions will result in a loss of signal below about 200±5 nm, which will make accurate secondary structure estimates more difficult, but generally won't interfere with the characteristic alpha-helical peaks at 208 and 222 or the beta-sheet peak at 218.
  • DMSO and formamides, which absorb strongly in the far UV region are not compatible with CD.
  • Imidazole absorbs strongly in the far UV and should be avoided.