Fluorescence spectroscopy in plate reader and cuvette
Coriolis Pharma is specialized in providing contract laboratory services related to protein characterization, including intrinsic and extrinsic fluorescence spectroscopy methods for protein conformation analysis.
Intrinsic fluorescence of proteins in solution is commonly measured in order to detect changes in protein conformation, e.g., due to different solution conditions (pH, excipients, etc…), elevated temperature and storage. Upon excitation at 280 nm, a protein’s fluorescence signal arises primarily from the presence of tryptophan and tyrosine residues. Selective excitation of tryptophan residues, e.g., at 295 nm, is also possible and commonly applied to proteins, because tryptophan fluorescence is particularly sensitive to subtle conformational changes. Tyrosine and tryptophan residues are typically buried in the core of a folded protein (not solvent exposed). Partial unfolding and aggregation lead to changes in the local environment of these residues (e.g., solvent exposure), leading to changes in fluorescence intensity and emission maximum, amongst others.
Extrinsic fluorescence can also be used to detect changes in protein structure and the formation of aggregates. Various fluorescent dyes are available, such as Nile Red, ANS and Bis-ANS, which can be used as hydrophobic probes. If a protein unfolds or aggregates, e.g., due to destabilizing solution conditions or increased temperature, the dye binds to hydrophobic patches on the protein (aggregates) and fluoresces with much greater intensity than free dye in solution.
Coriolis Pharma has three different fluorimeters:
The Tecan Safire2 plate reader and the Analytik Jena qTower2. Both instruments offer high-throughput screening with 96-well plate formats. The Tecan Safire2 is ideal for monitoring differences in intrinsic fluorescence due to different solution conditions.
The qTower2 is ideal for monitoring changes in extrinsic fluorescence due to different solution conditions and has the added functionality of temperature control, which allows the apparent melting temperature (Tm) to be determined by differential scanning fluorimetry (DSF).
In addition we run a cuvette based Jasco FP 8500 fluorimeter, suited for intrinsic and extrinsic fluorescence measurements.
Monitoring changes in extrinsic or intrinsic fluorescence has advantages and disadvantages and our team of expert scientists can advise about what is most appropriate for a given project.