National Hellenic Research Foundation (N.H.R.F.)  |   Fluorescence spectroscopy

Fluorescence spectroscopy is a versatile technique frequently used for the analysis of the tertiary structure of proteins. Most proteins exhibit intrinsic fluorescence, predominantly derived from tryptophan and tyrosine residues. Changes in protein conformation caused by different solution conditions (pH, excipients, etc.), elevated temperature, storage and/or interaction with other biomacromolecules can be detected by means of fluorescence measurements. This is due to the fact that tryptophan fluorescence (selective excitation at 295 nm) is particularly sensitive to the local environment of the residue. Thus, any conformational change (e.g. unfolding, aggregation) leads to changes in fluorescence intensity and/or emission maximum. Apart from intrinsic fluorescence, the use of suitable fluorophores or dyes (such as Nile red, ANS, FITC, pyrene) is also available in order to probe protein conformational changes.

National Hellenic Research Foundation (N.H.R.F.)
LOCATION:   Athens, Greece |
RELEVANT STAFF:   Nikos Tagmatarchis

At the Theoretical and Physical Chemistry Institute (TPCI) of NHRF a variety of spectroscopic techniques is available. As far as fluorescence spectroscopy is concerned the spectrometer available through the Instruct-EL hub, is a time-resolved fluorescence system (NanoLog, Horiba-Jobin Yvon) with 3 detectors: steady-state & time-resolved fluorescence / 2D NIR photoluminescence map capability together with multiple laser lines and cell temperature control. Fluorescence spectroscopic services involve conformational studies of proteins (native structure), as well as the investigation of structural changes (unfolding, refolding, aggregation) caused by changes in solution conditions or interactions with ligands/macromolecules, either at steady state or time resolved and kinetics experiments. Moreover, the specific instrumentation includes a reflectance apparatus, suited for measurements of thin films and surfaces.

Varioskan Flash by Thermo Fisher Scientific
TYPE:   Microplate reader Vis/UV/Fluorescence/ Luminescence
LOCATION:   Institute of Chemical Biology- Laboratory of Structural Biology / Instruct-EL hub
DESCRIPTION:   Microplate reader Vis/UV/Fluorescence/ Luminescence
FUNDER:   FP7-REGPOT-2009-1, No245866, "ARCADE"
Nanolog by Horiba-Jobin Yvon
TYPE:   Fluorosescence spectrometer
LOCATION:   Theoretical and Physical Chemistry Institute / Instruct-EL hub
DESCRIPTION:   Protein structure determination and studies on physicochemical properties in protein aqueous solutions
FUNDER:   EU-Capacities
In the last ten years, TPCI personnel has acquired a well-established experience in fluorescence spectroscopy and its use for conformational studies of proteins under various experimental conditions.