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. 2023:2652:199-213.
doi: 10.1007/978-1-0716-3147-8_11.

Screening of Buffers and Additives for Protein Stabilization by Thermal Shift Assay: A Practical Approach

Filipa S S Engrola  1   2 , João Paquete-Ferreira  1   2 , Teresa Santos-Silva  1   2 , Márcia A S Correia  3   4 , Francisco Leisico  5   6   7 , Marino F A Santos  8   9
Affiliations

Screening of Buffers and Additives for Protein Stabilization by Thermal Shift Assay: A Practical Approach

Filipa S S Engrola et al. Methods Mol Biol. 2023.

Abstract

Thermal shift assay (TSA), also commonly designed by differential scanning fluorimetry (DSF) or ThermoFluor, is a technique relatively easy to implement and perform, useful in a myriad of applications. In addition to versatility, it is also rather inexpensive, making it suitable for high-throughput approaches. TSA uses a fluorescent dye to monitor the thermal denaturation of the protein under study and determine its melting temperature (Tm). One of its main applications is to identify the best buffers and additives that enhance protein stability.Understanding the TSA operating mode and the main methodological steps is a central key to designing effective experiments and retrieving meaningful conclusions. This chapter intends to present a straightforward TSA protocol, with different troubleshooting tips, to screen effective protein stabilizers such as buffers and additives, as well as data treatment and analysis. TSA results provide conditions in which the protein of interest is stable and therefore suitable to carry out further biophysical and structural characterization.

Keywords: Additive screening; Buffer screening; Protein thermostability; Structural and biophysical protein characterization; Thermal shift assay (TSA).

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