Circular Dichroism - CD

 
 

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Instrument:


The JASCO J-810 spectropolarimeter equipped with a Peak Instruments N2-generator is located in the Michael Swann building, room 3.05.



Please click here for individual platform/resource costings.

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Features of the JASCO J-810 instrument:


The J-810 is capable of recording CD spectra from 190 – 800 nm. The sample compartment has a Peltier temperature controller and so CD spectra can be collected as a function of T as well as a function of time. The instrument is provided wit ha comprehensive software package that will convert the raw signal into ellipticity. Estimates of secondary structure can be obtained by submitting the spectra to the DICHROWEB website.


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What is Circular Dichroism?

Circular dichroism, CD, refers the different absorbance of left and right circularly polarised light. It is useful to probe chiral centres in molecules and, for proteins, it can be used:

  1. to obtain estimates of the backbone conformation.

  2. to obtain information about the environment of the aromatic residues Trp and Tyr.

  3. to measure ligand binding provided there is a detectable change in the spectrum on association.  The size range of molecules (radius) detectable is 0.3 nm (corresponding to ca. 380 Da. protein) to 0.5 microns.

As the phenomenon is recorded as the difference in two signals, it is easy to produce nonsense if the protein concentration is too high, or if some other component absorbs strongly. For proteins, the main chain conformation is defined by the amide absorption of the peptide bonds which are intense at short wavelengths, so either very dilute solutions or very short path lengths are needed. Because one can follow the change in spectrum, or at a fixed wavelength with time, temperature or ligand concentration experiments can be designed to show unfolding or refolding, as well as determination of the association constant.

Protein samples should be free of dust and other particulate matter, properly buffered in a suitable buffer and of well-defined concentration. For work in the region between 190 and 220 nm, it is important to remember that some ions (e.g. chloride absorb strongly below 200nm so high concentrations should be avoided. The same obviously applies to the buffers – phosphate and borate are suitable. Concentrations in the range 0.5 – 2 mg/mL with a pathlength of 0.02 to 0.1 cm are generally suitable for the 190–250 nm range where the same concentration in a 1 cm cuvette is alright for the aromatic region. Note that the sample cells need to be specially checked for their suitability for CD measurements. Hellma and Starna both provide this service.

An excellent reference giving fuller details of the technique is Kelly et al. (2005).

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contacts:


Facilities Manager:

Dr. Martin Wear

Rm. Swann 3.20

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: martin.wear@ed.ac.uk


Snr.
Protein Technologist:

Dr. Liz Blackburn

Rm. Swann 3.19

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: E.A.Blackburn@ed.ac.uk


Snr. Protein Technologist:

Dr. Matt Nowicki

Rm. Swann 3.19

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: matthew.nowicki@ed.ac.uk


Facilities:


CTCB-Home

Protein Production Biophysical Characterisation

In Silico Screening

Training


Links:


On-line Booking

Core Column Library


Access Charges/Costs:


Access Charges/Costs

 

Location:

The University of Edinburgh,

Level 3 Michael Swann Building,

King’s Buildings,

Mayfield Rd.,

EH9 3JR,

UK


Lab 3.10

CD - JASCO J-810