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.
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.
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.