Jasco J810 Circular Dichroism Spectrometer

The Jasco J810 CD spectrometer is commonly used in the UV region of the spectrum to determine protein secondary structure1. The upper scan limit is 900 nm so it is also possible to measure CD spectra of proteins containing cofactors (e.g., a heme group, or a flavin) that may be CD active in non-isotropic environments within a protein. This CD spectrometer is equipped with a computer-controlled Peltier device (3-90 °C) and a two-syringe titrator to asses thermal/chemical stabilities of proteins/ polypeptides or to monitor ligand binding.

First-time users please contact the hub manager to arrange for training on the use of this instrument. Qualified users can book instrument time online with the user name cd_UBC. On the day(s) of your experiment(s), you will need to bring with you the following supplies :

 

 

  • cleared samples (centrifuged or filtered)1
  • sample buffer2
  • oxygen-free nitrogen gas3
  • quartz cuvette(s)4
  • pipetters and pipet tips
  • Parafilm
  • cleaning solutions (water, detergents, acid, alcohol, etc.)
  • clean wipes
  • USB thumbdrive(s)
  • printer paper

1To determine the secondary structure of a protein users need to collect spectra between 190 and at least 260 nm. Prepare ~250 mL of each protein sample at ~ 5 mM protein. If the absorbance of such a solution is too high, the sample may require additional dilution to optimize the signal quality. Excessive optical densities (> 650 V gain on the detector) result in the acquisition of low quality spectra (i.e., poor signal-to-noise ratios). A gain greater than 800 V is harmful to the detector.

2Use low concentrations (10 mM or less) of high purity buffers and salts (e.g., sulfonic acids, phosphate, Tris, or acetate buffers); contaminants with high absorptivity will degrade signal quality even when using short pathlength cuvettes. It is also recommended that you keep [chloride] low if data are required below 200 nm. Deoxygenating buffers by flushing the buffer with nitrogen gas immediately before use may also help reduce detector gain. Do not use either HEPES buffer or nitrate salts. Assess their suitablility of reducing or denaturing agents in your experiment before wasting time and/or valuable samples.

3Nitrogen gas is needed to purge the light path through the spectrometer. Insufficient flushing of the instrument leads not only to high absorbances but also to the ionization of residual oxygen in the lightpath to ozone, which is corrosive and therefore harmful for the instrument optical components. It is up to spectrometer users to supply the nitrogen for their work so please order a liquid nitrogen dewar or from a T-cylinder of pre-purified, compressed gas pp4.8 from Praxair.

4The Peltier cuvette holder is designed for 1 x 1 cm cuvettes rather than the traditional cylindrical cuvettes. Shorter pathlength cuvettes can be fitted with a suitable "wedge". For thermal denaturations, 1 mm pathlength, capped, quartz cuvettes (e.g., Hellma 110-1-40) are recommended for efficient heat exchange.

For practical considerations and examples, consult the three Nature Protocols papers by Norma J. Greenfield :

To learn more about circular dichroism theory, consult also :

  • Circular dichroism and the conformational analysis of biomolecules, edited by Gerald D. Fasman (Woodward library QD473.C573 1996)
  • Circular dichroism. Principles and applications, edited by Berova, Nakanishi, and Woody

A number of applications and resources are available on the web to determine the secondary structure of protein samples empirically. These sites feature the programs SOMCD, K2d, CD1/2, DICROPROT and CDpro.

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