CD sample preparation

Some general information on CD of proteins can be downloaded here as a pdf file.
Choice of Buffer: Many common buffer components absorb strongly, particularly at shorter wavelengths, and can mask signals of interest. In general, try to keep buffer concentrations as low as possible and observe the following:
  • Use UV-grade solvents.
  • Avoid using water that has been stored in polyethylene bottles for a long time as it may contain dissolved polymer additives.
  • 10mM potassium phosphate is a good choice for most work. Low concentrations of perchlorate, Tris, sodium phosphate and borate are also reasonably transparent.
  • SO42- or F- is preferred counter ion as Cl- has a strong UV absorbance at low wavelengths.
  • DTT, BME, or EDTA can be present at low concentrations (≤1mM).
  • Imidazole absorbs strongly in the far UV. Millimolar concentrations of imidazole will swamp your micromolar protein signal.
  • Buffers can contain up to 20% glycerol, but measurements can only be made to 200 nm at this concentration.
  • SDS, Chaps and octylglucoside are reasonably transparent detergents. Avoid Triton detergents as they tend to oxidize rapidly and form UV-absorbing materials.
  • The pH of Tris is highly dependent on temperature, which makes it a poor choice for thermal melts.
  • For denaturant melts, be sure to use ultrapure spectral grade urea or guanidine.
Approximate wavelength cutoffs (nm) of various solvents/buffers:
  1 cm cell 1 mm cell
Distilled Water 185 <185
100mM Ammonium Citrate   220
150mM Ammonium Sulfate   190
100mM Mes   205
100mM Pipes   215
100mM Sodium Chloride   195
10mM Sodium Phosphate   <185
100mM Sodium Phosphate   190
PBS (phosphate buffered saline)   200
100mM Tris-HCl   200
Acetonitrile   185
DMSO 264 252
Ethanol 210 195
Hexafluoroisopropanol   <185
Methanol 210 195
Trifluoroethanol   <185
6M GdnHCl (Sigma high spectral grade) 218  
4M GdnHCl   210
4M Urea   210

Filtration: Filtering your samples and buffer through a 0.2µ or 0.45µ filter will remove dust, aggregated protein and other particles that interfere with CD measurements.

Cell Pathlength: The CSB owns both standard and semi-micro cells that you can use.

  • Smaller pathlength cells (1mm) decrease solvent absorbance and are used for far-UV measurements.
    • A typical concentration for a 1 mm cell is ~0.1 mg/mL
    • Minimum volume ~150µL
  • Longer pathlength cells (1cm) are used for near-UV measurements since the signals are usually weak.
    • Near-UV measurements typically require high concentrations, ~1-2 mg/mL
    • Minimum volume of the standard cell is ~2mL
    • The CSB also has two masked cells with minimum volumes of ~500µL and ~160µL

CD Operational Notes: