CD - Instruct-CZ

Circular dichroism spectroscopy measures differences in the absorption on left and right - handed polarised lite that arrise from the structural assymetry (presence in chiral atoms in the molecule). CD spectroscopy is used to investigate the secondary structure of proteins α-helices, β-strands and randome coils can be identified in the fare UV region where thay give rise to characteristic shape and magnitude of the spectrum.

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CD - Instruct-CZ

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User Guide

CD spectropolarimeter Jasco J-815 - This instrument can measure: circular dichroism, fluorescence, total fluorescence, linear dichroism, magnetic dichroism, optical rotation dispersion, and stopped flow circular dichroism, fluorescence and absorbance. Circular dichroism and fluorescence data can be acquired simultaneously. Wavelength range 185 – 900 nm. All the measurements on JASCO-J815 are done in 1 – 10 mm cells according to buffer composition and protein concentration.

Accessories:
  • Peltier temperature control
  • Monochromator for fluorescence
  • Bio-Logic: SFM-20; two channel stopped-flow setup
Circular dichroism can be used:
  • for the determination of protein folding
  • to characterize protein`s secondary structure
  • to detect the changes in structure upon mutagenesis
  • to study conformational stability of proteins (pH stability, denaturant stability, temperature, buffers addition of stabilizers) or
  • to detect the changes in the conformation of a protein upon protein-protein interaction
Data collection:
  • Wavelength scanning:
    • Continuous scan: running average method offering high speed measurements
    • Step Scan: discrete wavelengths and response time to optimize signals
    • Auto-scan: based on step scan but offering a range of response times to speed data acquisition
  • Time scan
    • Fixed wavelength time scan for chemical denaturation and stopped-flow experiments
  • Temperature scan
    • Fixed wavelength for CD vs. Temperature thermal ramping
    • Pre-set temperatures with equilibration times for spectral scanning
    • 3 Dimensional display of CD vs. Wavelength vs. Temperature or Time
Data processing (possibility to train the people how to analyse the data):
  • JASCO's new - Spectra Manager system
  • DICHROweb
  • K2D3
CD Spectroscopy requirements:

Far-UV CD spectra (secondary structure measurement) require between 300 µl - 700 µl of ~ 0.1 – 0.5 mg/ml protein solution, in any buffer, which does not show a high absorbance in this region of the spectrum. Substances not optimal for CD: DTT or stabilizing salts (high concentrations only), imidazole, Triton X-100.