Skip to main content
SpringerLink
Log in

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

  • Protocol
  • First Online:
Advanced Methods in Structural Biology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2652))

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Chua EYD, Mendez JH, Rapp M, Ilca SL et al (2022) Better, faster, cheaper: recent advances in cryo–electron microscopy. Annu Rev Biochem 91:1–32

    Article  PubMed  Google Scholar 

  2. Powell HR (2019) From then till now: changing data collection methods in single crystal X-ray crystallography since 1912. Crystallogr Rev 25:264–294

    Article  CAS  Google Scholar 

  3. Magnusson AO, Szekrenyi A, Joosten H et al (2019) nanoDSF as screening tool for enzyme libraries and biotechnology development. FEBS J 286:184–204

    Article  CAS  PubMed  Google Scholar 

  4. Wen J, Lord H, Knutson N et al (2020) Nano differential scanning fluorimetry for comparability studies of therapeutic proteins. Anal Biochem 593:113581

    Article  CAS  PubMed  Google Scholar 

  5. Forneris F, Orru R, Bonivento D et al (2009) Thermo FAD, a Thermofluor® -adapted flavin ad hoc detection system for protein folding and ligand binding. FEBS J 276:2833–2840

    Article  CAS  PubMed  Google Scholar 

  6. Ericsson UB, Hallberg BM, DeTitta GT et al (2006) Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal Biochem 357:289–298

    Article  CAS  PubMed  Google Scholar 

  7. Senisterra G, Chau I, Vedadi M (2012) Thermal denaturation assays in chemical biology. Assay Drug Dev Technol 10:128–136

    Article  CAS  PubMed  Google Scholar 

  8. Simeonov A (2013) Recent developments in the use of differential scanning fluorometry in protein and small molecule discovery and characterization. Expert Opin Drug Discov 8:1071–1082

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Boivin S, Kozak S, Meijers R (2013) Optimization of protein purification and characterization using Thermofluor screens. Protein Expr Purif 91:192–206

    Article  CAS  PubMed  Google Scholar 

  10. Vivoli M, Novak HR, Littlechild JA et al (2014) Determination of protein-ligand interactions using differential scanning fluorimetry. J Vis Exp 91:e51809

    Google Scholar 

  11. Gao K, Oerlemans R, Groves MR (2020) Theory and applications of differential scanning fluorimetry in early-stage drug discovery. Biophys Rev 12:85–104

    Article  PubMed  PubMed Central  Google Scholar 

  12. Li X, Zhang C (2021) Using differential scanning fluorimetry (DSF) to detect ligand binding with purified protein. Methods Mol Biol 2213:183–186

    Article  CAS  PubMed  Google Scholar 

  13. Bischof JC, He X (2006) Thermal stability of proteins. Ann N Y Acad Sci 1066:12–33

    Article  Google Scholar 

  14. Miotto M, Olimpieri PP, di Rienzo L et al (2019) Insights on protein thermal stability: a graph representation of molecular interactions. Bioinformatics 35:2569–2577

    Article  CAS  PubMed  Google Scholar 

  15. Correia Cordeiro RS, Enoki J, Busch F et al (2018) Cloning and characterization of a new delta-specific l-leucine dioxygenase from Anabaena variabilis. J Biotechnol 284:68–74

    Article  CAS  PubMed  Google Scholar 

  16. de Benedetti S, Leogrande C, Castagna F et al (2022) Thermal shift assay as a tool to evaluate the release of breakdown peptides from cowpea β-vignin during seed germination. Molecules 27:277

    Article  PubMed  PubMed Central  Google Scholar 

  17. Vedadi M, Niesen FH, Allali-Hassani A et al (2006) Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc Natl Acad Sci 103(15835–15):840

    Google Scholar 

  18. Geders TW, Gustafson K, Finzel BC (2012) Use of differential scanning fluorimetry to optimize the purification and crystallization of PLP-dependent enzymes. Acta Crystallogr Sect F Struct Biol Cryst Commun 68:596–600

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Reinhard L, Mayerhofer H, Geerlof A et al (2013) Optimization of protein buffer cocktails using Thermofluor. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:209–214

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kozak S, Lercher L, Karanth MN et al (2016) Optimization of protein samples for NMR using thermal shift assays. J Biomol NMR 64:281–289

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Pantoliano MW, Petrella EC, Kwasnoski JD et al (2001) High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen 6:429–440

    Article  CAS  PubMed  Google Scholar 

  22. Santos SP, Bandeiras TM, Pinto AF et al (2012) Thermofluor-based optimization strategy for the stabilization and crystallization of Campylobacter jejuni desulforubrerythrin. Protein Expr Purif 81:193–200

    Article  CAS  PubMed  Google Scholar 

  23. Barroca-Ferreira J, Cruz-Vicente P, Santos MFA et al (2021) Enhanced stability of detergent-free human native STEAP1 protein from neoplastic prostate cancer cells upon an innovative isolation procedure. Int J Mol Sci 22:10012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Gradl S, Steuber H, Weiske J et al (2021) Discovery of the SMYD3 inhibitor BAY-6035 using thermal shift assay (TSA)-based high-throughput screening. SLAS Discov 26:947–960

    Article  CAS  PubMed  Google Scholar 

  25. Tatum NJ, Liebeschuetz JW, Cole JC et al (2017) New active leads for tuberculosis booster drugs by structure-based drug discovery. Org Biomol Chem 15:10245–10255

    Article  CAS  PubMed  Google Scholar 

  26. Andreotti G, Monticelli M, Cubellis MV (2015) Looking for protein stabilizing drugs with thermal shift assay. Drug Test Anal 7:831–834

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Ramos J, Muthukumaran J, Freire F et al (2019) Shedding light on the interaction of human anti-apoptotic Bcl-2 protein with ligands through biophysical and in silico studies. Int J Mol Sci 20:860

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Rosa N, Ristic M, Seabrook SA et al (2015) Meltdown: a tool to help in the interpretation of thermal melt curves acquired by differential scanning fluorimetry. J Biomol Screen 20:898–905

    Article  CAS  PubMed  Google Scholar 

  29. Martin-Malpartida P, Hausvik E, Underhaug J et al (2022) HTSDSF explorer, a novel tool to analyze high-throughput DSF screenings. J Mol Biol 434:167372

    Article  CAS  PubMed  Google Scholar 

  30. Bai N, Roder H, Dickson A et al (2019) Isothermal analysis of ThermoFluor data can readily provide quantitative binding affinities. Sci Rep 9:2650

    Article  PubMed  PubMed Central  Google Scholar 

  31. Bui-Le L, Clarke CJ, Bröhl A et al (2020) Revealing the complexity of ionic liquid–protein interactions through a multi-technique investigation. Commun Chem 3:55

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kumar A, Venkatesu P (2018) Innovative aspects of protein stability in ionic liquid mixtures. Biophys Rev 10:841–846

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Schindl A, Hagen ML, Muzammal S et al (2019) Proteins in ionic liquids: reactions, applications, and futures. Front Chem 7:347

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Ehrhardt MKG, Warring SL, Gerth ML (2018) Screening chemoreceptor-ligand interactions by high-throughput thermal-shift assays. Methods Mol Biol 1729:281–290

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

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 Fundação para a Ciência e Tecnologia (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.

Author information

Authors and Affiliations

  1. Associate Laboratory i4HB – Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, Caparica, Portugal

    Filipa S. S. Engrola, João Paquete-Ferreira, Teresa Santos-Silva, Márcia A. S. Correia, Francisco Leisico & Marino F. A. Santos

  2. UCIBIO, Applied Molecular Biosciences Unit, Chemistry Department, NOVA School of Science and Technology, Universidade NOVA de Lisboa, Caparica, Portugal

    Filipa S. S. Engrola, João Paquete-Ferreira, Teresa Santos-Silva, Márcia A. S. Correia, Francisco Leisico & Marino F. A. Santos

  3. Institut de Biologie Structurale, UMR 5075, University Grenoble Alpes, CNRS, CEA, Grenoble, France

    Francisco Leisico

Authors
  1. Filipa S. S. Engrola
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. João Paquete-Ferreira
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Teresa Santos-Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Márcia A. S. Correia
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Francisco Leisico
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marino F. A. Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Márcia A. S. Correia , Francisco Leisico or Marino F. A. Santos .

Editor information

Editors and Affiliations

  1. Universidade da Beira Interior, CICS-UBI, Covilhã, Portugal

    Ângela Sousa

  2. University of Beira Interior, Covilhã, Portugal

    Luis Passarinha

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Engrola, F.S.S., Paquete-Ferreira, J., Santos-Silva, T., Correia, M.A.S., Leisico, F., Santos, M.F.A. (2023). Screening of Buffers and Additives for Protein Stabilization by Thermal Shift Assay: A Practical Approach. In: Sousa, Â., Passarinha, L. (eds) Advanced Methods in Structural Biology. Methods in Molecular Biology, vol 2652. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3147-8_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3147-8_11

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3146-1

  • Online ISBN: 978-1-0716-3147-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation