Taking circular dichroism spectra

Harms Lab Wiki > Protein Biochemistry > > Taking circular dichroism spectra

Circular dichroism can be used to study the conformation of a protein in solution.

Some things to keep in mind:

  • Your buffer choice matters.  CD requires taking the difference between two absorbance measurements.  If your buffer absorbs strongly at a given wavelength, very little light will make it through your sample, leading to noise.  (In the extreme, no light gets through your sample and you see nothing at all).  Unfortunately, most common buffers, as well as chloride ions, absorb in the far UV (250 nm and down).  The exception is phosphate.  Using 25 mM phosphate + 100 mM NaCl will allow you to get to ~200 nm.  If you need to go below this, you can lower the ionic strength or, if that’s not possible, use sodium flouride instead of sodium chloride.  (Note that there are particular considerations if you’re taking a spectrum using divalent metals).
  • Your pathlength matters.  Most CD work is done with either a 1 mm or 10 mm pathlength.  The choice of pathlength is a compromise between increasing signal (10 mm is better) and making sure enough photons make it through to get an accurate measurement (1 mm is better).  For far-UV CD (< 250 nm), a 1 mm cuvette is generally better; for near-UV and visible CD (> 250 nm), a 10 mm cuvette is generally better).
  • Your protein concentration matters.  Comparisons between CD spectra require normalizing to the concentration of the sample, so the concentration should be determined with as high an accuracy as possible.  Use the best practices for measuring protein concentration by absorbance.

Sample preparation

Stub.

Turning on the instrument

  1. Turn on the nitrogen flow.  This is done by turning the green [xxx] knob on top of the tank and then the small knob near the computer so the float reads “20.”  You should not have to adjust the regulator.  (NEVER RUN THE SYSTEM WITHOUT N2!  The Xenon lamp pumps out UV, which converts oxygen into ozone, which etches the mirrors along the optical path.  Flooding with N2 minimizes the damage to the optics).
  2. Turn on the instrument itself (rocker switch on front of system).
  3. Turn on the water bath (if you’re using temperature control).
  4. Turn on the computer and open the “SpectraManager” software.
  5. After the N2 has been running for >15 minutes, turn on the lamp.  Select [wavelength spectra] and click the lamp icon above the graph.  You should hear a click, followed by a fan.
  6. Let the lamp warm up for ~1 hour prior to use.

Taking a measurement

Stub. This describes taking a spectrum, with notes about what the settings mean etc.  This will be relevant to other collection types as well.

Shutting down the instrument

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Data processing

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