Real-time protein unfolding: a method for determining the kinetics of native protein denaturation using a quantitative real-time thermocycler

Biggar, K. K., Dawson, N. J. and Storey, K. B. (2012) Real-time protein unfolding: a method for determining the kinetics of native protein denaturation using a quantitative real-time thermocycler. BioTechniques, 53(4), pp. 231-238. (doi: 10.2144/0000113922) (PMID:23046506)

Abstract

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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Dawson, Dr Neal
• Authors: Biggar, K. K., Dawson, N. J., and Storey, K. B.
• College/School: College of Medical Veterinary and Life Sciences > School of Biodiversity, One Health & Veterinary Medicine
• Journal Name: BioTechniques
• Publisher: Future Medicine Ltd.
• ISSN: 1940-9818
• ISSN (Online): 1940-9818