Location: Level 1 South, Room 120
Model: Agilent Technologies Cary Eclipse
Fluorescence measurements are useful for the study of such diverse processes as enzyme kinetics, biomolecular interactions and protein structure and folding.
Types of experiments that can be performed on the MIF instrument include:
- Emission wavelength scans, which can potentially provide structural insights into a protein, as the emission spectrum of a particular substituent (e.g., tryptophan or an added fluorophore) can be sensitive to its immediate environment. Such measurements can be extended with the addition of non-fluorescent molecules that quench the fluorescence from a macromolecule, thus probing the accessibility of the fluorescent substituent.
- The modulation of emission intensity and/or wavelength by the addition of either fluorescent or non-fluorescent molecules to measure interaction, as used in Forster resonance energy transfer (FRET) experiments. FRET measurements yield information on the proximity of one fluorescent molecule to another, providing information on conformation or binding.
- Fluorescence anisotropy, which can be used to measure the binding constants and kinetics of reactions that cause a change in the rotational time of the molecules. If the fluorophore is bound to a small molecule, the rate at which it tumbles decreases significantly when the molecule is bound tightly to a protein. If the fluorophore is attached to the larger protein in a binding pair, the difference in polarization between bound and unbound states is smaller. The degree of binding is calculated by using the difference in anisotropy of the partially bound, free and fully bound states.
For users wishing to gain a deeper understanding of fluorescence spectroscopy, the recommended text is:
- Principles of Fluorescence Spectroscopy, by Joseph R. Lacowicz | Springer Publishing, ISBN: 978-0-387-46312-4