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.

Additional Metadata
Keywords Fluorescence, Hexokinase, Lysozyme, Protein stability, QRT-PCR, SYPRO orange, Urea
Persistent URL dx.doi.org/10.2144/0000113922
Journal BioTechniques
Citation
Biggar, K.K, Dawson, N.J. (Neal J.), & Storey, K. (2012). Real-time protein unfolding: A method for determining the kinetics of native protein denaturation using a quantitative real-time thermocycler. BioTechniques, 53(4), 231–238. doi:10.2144/0000113922