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Protein stability can be monitored by many different techniques. However, these protocols are often lengthy, consume large amounts of protein, and require expensive and specialized instruments. Here we present a new protocol to analyze protein unfolding kinetics using a quantified real-time thermocycler. This technique enables the analysis of a wide range of denaturants (and their interactions with temperature change) on protein stability in a multi-well platform, where samples can be run in parallel under virtually identical conditions and with highly sensitive detection. Using this set-up, researchers can evaluate the half-maximal rate of protein denaturation (Knd), maximum rate of denaturation (Dmax), and the cooperativity of individual denaturants in protein unfolding (µ-coefficient). Both lysozyme and hexokinase are used as model proteins and urea as a model denaturant to illustrate this new method and the kinetics of protein unfolding that it provides. Overall, this method allows the researcher to explore a large number of denaturants, at either constant or variable temperatures, within the same assay, providing estimates of denaturation kinetics that have been previously inaccessible.