High-throughput, kinetic protein unfolding assays developed on the POLARstar Omega microplate reader
Determining the protein unfolding constant is a basic process in the characterization of protein dynamics. Such parameters help to understand protein stability and are the basis e.g. for protein binding specificity investigations.
In this application note we show how to determine the unfolding constant with the help of a fluorescence measurement. The three dimensional folded form of a protein exhibits tryptophan residues at the protein surface resulting in a certain fluorescence value. After addition of a denaturation solution protein unfolding will start. The result is a decrease of fluorescence.
Some proteins show a very fast unfolding behavior. For these protein samples it is necessary to inject the denaturation solution and measure the fluorescence signal at the same time. The POLARstar® Omega microplate reader from BMG LABTECH was used to develop a protein unfolding assay because of its capabilities to measure at time of injection followed by high frequency readings (measurement at data point every 120 ms).