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Center for Structural Biology

CD Sample Preparation

Some general information on CD of proteins can be downloaded 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.
  • 10 mM 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 (≤1 mM).
  • 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
100 mM Ammonium Citrate 220
150 mM Ammonium Sulfate 190
100 mM MES 205
100 mM PIPES 215
100 mM Sodium Chloride 195
10 mM Sodium Phosphate <185
100 mM Sodium Phosphate 190
PBS (phosphate buffered saline) 200
100 mM Tris-HCl 200
Acetonitrile 185
DMSO 264 252
Ethanol 210 195
Hexafluoroisopropanol <185
Methanol 210 195
Trifluoroethanol <185
6 M GdnHCl (Sigma high spectral grade) 218
4 M GdnHCl 210
4 M Urea 210

 

Filtration

Filtering your samples and buffer through a 0.2 µm or 0.45 µm 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 (1 mm) 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 ~150 µL
  • 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 ~2 mL
    • The CSB also has two masked cells with minimum volumes of ~500 µL and ~160 µL