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PLoS One. 2016 Jan 8;11(1):e0146232. doi: 10.1371/journal.pone.0146232. eCollection 2016.

Development and Application of a High Throughput Protein Unfolding Kinetic Assay.

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Department of Physical Sciences, Eastern New Mexico University, Portales, New Mexico, United States of America.
NMRFAM, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.


The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses.

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