Jasco J-815 Circular Dichroism (CD) Spectropolarimeter
Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light that arise due to structural asymmetry. The absence of regular structure results in zero CD intensity, while an ordered structure results in a spectrum which can contain both positive and negative signals.
CD can be used for:
- Determination if a protein is folded
- Characterization of its secondary structure (α-helix, β-sheet)
- Detection of changes in structure upon mutagenesis
- Studying conformational stability of proteins:
- pH stability
- denaturant stability (urea, guadinium hydrochloride)
- temperature
- buffers
- addition of stabilizers
- Detection of Changes in the conformation of a protein upon protein:protein interaction
Characterization of its secondary structure is probably the most used CD spectroscopy application. Secondary structure can be identified in the "far-UV" spectral region (190-250 nm). The protein peptide bond is the chromophore, and it is possible to detect a signal if the protein is in a specific secondary structural conformation (α-helix, β-sheet).
Alpha-helix, beta-sheet, and random coil structures each give rise to a characteristic shape and magnitude of CD spectrum. Like all spectroscopic techniques, the CD signal reflects an average of the entire molecular population.
CD Spectroscopy requirements:
Far-UV CD spectra require between 100 µl- 700 µl of ~ 3-10 µM protein solution, in any buffer which does not have a high absorbance in this region of the spectrum.
Substances not optimal for CD: DTT (high concentrations only), imidazole, Triton
X-100.
Accessories:
- Pelitier temperature control.
- Automated Titration Systems allow long term kinetic, chemical denaturation, and ligand binding studies in a completely unattended mode.
- Bio-Logic: SFM-20; two channel stop-flow setup.