Sorenson, Alanna E., and Schaeffer, Patrick M. (2020) High-throughput differential scanning fluorimetry of GFP-tagged proteins. In: Labrou, Nikolaos E., (ed.) Targeting Enzymes for Pharmaceutical Development. Methods in Molecular Biology, 2089 . Springer, New York, NY, USA, pp. 69-85.

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Abstract

Differential scanning fluorimetry is useful for a wide variety of applications including characterization of protein function, structure–activity relationships, drug screening, and optimization of buffer conditions for protein purification, enzyme activity, and crystallization. A limitation of classic differential scanning fluorimetry is its reliance on highly purified protein samples. This limitation is overcome through differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP). DSF-GTP specifically measures the unfolding and aggregation of a target protein fused to GFP through its proximal perturbation effects on GFP fluorescence. As a result of this unique principle, DSF-GTP can specifically measure the thermal stability of a target protein in the presence of other proteins. Additionally, the GFP provides a unique in-assay quality control measure. Here, we describe the workflow, steps, and important considerations for executing a DSF-GTP experiment in a 96-well plate format.

Item ID:	65792
Item Type:	Book Chapter (Scholarly Work)
ISBN:	978-1-0716-0163-1
ISSN:	1064-3745
Keywords:	Fluorimetry, High-throughput screening, Thermal shift assay, Green fluorescent protein,Ligand binding, Enzyme inhibitors, Selective protein unfolding, Drug discovery
Copyright Information:	© Springer Science+Business Media, LLC 2020
Date Deposited:	15 Mar 2021 02:36
FoR Codes:	31 BIOLOGICAL SCIENCES > 3101 Biochemistry and cell biology > 310109 Proteomics and intermolecular interactions (excl. medical proteomics) @ 100%
SEO Codes:	97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 100%
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