TY - CHAP
T1 - Screening of Buffers and Additives for Protein Stabilization by Thermal Shift Assay
T2 - A Practical Approach
AU - Engrola, Filipa S. S.
AU - Paquete-Ferreira, João
AU - Santos-Silva, Teresa
AU - Correia, Márcia A. S.
AU - Leisico, Francisco
AU - Santos, Marino F. A.
N1 - Funding Information:
This work was supported by the Applied Molecular Biosciences Unit UCIBIO (UIDB/04378/2020 and UIDP/04378/2020) and the Associate Laboratory Institute for Health and Bioeconomy–i4HB (project LA/P/0140/2020) which are financed by National Funds from Fundac¸ão para a Ciência e Tecno-logia (FCT). Filipa S. S. Engrola (UID/04378/2020) acknowledges her PhD Fellowship to UCIBIO and FCT; João Paquete-Ferreira (2020.08580.BD) and Francisco Leisico (PD/BD/ 105737/2014) acknowledge their PhD Fellowships to FCT. The authors would like to thank the current and past members of the Macromolecular Crystallography Lab (UCIBIO, FCT-NOVA) for their valuable suggestions over the years, contributing to the development of this easy-to-follow protocol. Filipe Freire and João Ramos are acknowledged for their critical reading of some sections of the presented chapter.
Publisher Copyright:
© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
KW - Additive screening
KW - Buffer screening
KW - Protein thermostability
KW - Structural and biophysical protein characterization
KW - Thermal shift assay (TSA)
UR - http://www.scopus.com/inward/record.url?scp=85153687320&partnerID=8YFLogxK
U2 - 10.1007/978-1-0716-3147-8_11
DO - 10.1007/978-1-0716-3147-8_11
M3 - Chapter
C2 - 37093477
AN - SCOPUS:85153687320
SN - 978-1-0716-3146-1
T3 - Methods in Molecular Biology
SP - 199
EP - 213
BT - Advanced Methods in Structural Biology
A2 - Sousa, Ângela
A2 - Passarinha, Luís
PB - Humana Press
CY - New York
ER -