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Circular Dichroism Spectrometer
(CD)

Circular dichroism measures the interaction of circularly polarized light with molecules.  Circularly polarized light interacts equally with non-chiral molecules so non-chiral molecules are not measured.  As the circularly polarized light passes through an optically active substance, its two circularly polarized components travel at different speeds and are absorbed in differing degrees.  The Jasco J-710 circular dichroism spectrometer measures this difference in the absorption of left-handed polarized light versus right-handed polarized light that results from structural asymmetry. 

CD spectroscopy is a standard method for measuring conformation in chiral molecules.  CD spectra between 260 and approximately 180 nm can be analyzed for secondary structural elements: alpha helix, parallel and anti-parallel beta sheet, turn, and other. The absence of regular structure results in zero CD intensity, while an ordered structure reveals a spectrum that can contain both positive and negative signals.

Applications

  • Characterization of protein secondary and tertiary structure
  • Studying the conformational stability of a protein at varying temperature, pH or denaturant concentrations
  • Determination of the effects of protein-protein interactions on conformation
  • Comparing the structures of protein vs. mutants or proteins expressed in different systems

Specifications

  • You do NOT need to bring liquid nitrogen.  The CD is attached to a nitrogen outlet in the small instrument room.

Buffer Requirements

  • Buffers that absorb in the region of interest should be avoided.
  • 10 mM sodium or potassium phosphate buffer is generally recommended for CD.  Tris or TEA can be used but should be pH'd with sulphuric or phosphoric acid,
  • Many buffers absorb at the shorter wavelengths where most of the structural information of interest is found.  AVOID chloride, citrates, MOPS, immidazole and DTT.
  • Also record a spectrum of the buffer alone before starting with samples to ensure the buffer absorbance is not a problem.

Sample Requirements

  • The sample should be as pure as possible since impurities will contribute to the the CD signal.
  • The sample concentration is determined by the pathlength of your cuvette.  A starting guideline is the sample should not exceed an absorbance of 0.9 OD over the wavelengths you want to scan.  (Take an asorbance scan of your sample through the wavelengths you plan to use in your experiment to ensure your sample does not exceed 0.9 OD at any wavelength.)  In a 1mm cuvette, a good starting point is 0.2 mg/ml for the 190-230nm range while 0.02 mg/ml would be used for a 1cm cuvette and 2mg/ml for a 0.1mm cuvette.
  • A smaller pathlength cuvette will decrease solvent absorbance to allow scanning down to lower wavelengths but will require a more concentrated sample.
  • Sample volume is determined by the cuvette used.  The sample must cover the light beam.

Supplies You Should Bring for Your Experiment

  • Cuvettes suitable for CD.  CD cuvettes are made of high-grade quartz that have been specially annealed to reduce strains in the quartz that can cause background noise.
  • Pipettes and tips
  • Kimwipes or lens paper
  • Your sample and buffer
  • A floppy or ZIP disc to save your data

Training, Assistance or Instrument Problems

If you need training or some assistance with your CD experiment, please contact Ben Ramirez PRIOR to scheduling instrument time.

If there is a problem with the instrument, please let Ben Ramirez know.  If you cannot find him in the facility, leave a note on his door in Room 112 or send an email. 

Manual and Articles

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