Abstract
Interactions between nucleic acids and proteins are driving gene expression programs and regulating the development of organisms. The binding affinities of transcription factors to their target sites are essential parameters to reveal their binding site occupancy and function in vivo. Microscale Thermophoresis (MST) is a rapid and precise method allowing for quantitative analysis of molecular interactions in solution on a microliter scale. The technique is based on the movement of molecules in temperature gradients, which is referred to as thermophoresis, and depends on molecule size, charge, and hydration shell. Since at least one of these parameters is typically affected upon binding of a ligand, the method can be used to analyze any kind of biomolecular interaction. This section provides a detailed protocol describing the analysis of DNA–protein interactions, using the transcription factor TTF-I as a model protein that recognizes a 10 bp long sequence motif.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Grummt I, Maier U, Ohrlein A, Hassouna N, Bachellerie JP (1985) Transcription of mouse rDNA terminates downstream of the 3′ end of 28S RNA and involves interaction of factors with repeated sequences in the 3′ spacer. Cell 43(3 Pt 2):801–810
Evers R, Grummt I (1995) Molecular coevolution of mammalian ribosomal gene terminator sequences and the transcription termination factor TTF-I. Proc Natl Acad Sci U S A 92(13):5827–5831
Langst G, Becker PB, Grummt I (1998) TTF-I determines the chromatin architecture of the active rDNA promoter. EMBO J 17(11):3135–3145. doi:10.1093/emboj/17.11.3135
Diermeier SD, Nemeth A, Rehli M, Grummt I, Langst G (2013) Chromatin-specific regulation of mammalian rDNA transcription by clustered TTF-I binding sites. PLoS Genet 9(9):e1003786. doi:10.1371/journal.pgen.1003786
Duhr S, Braun D (2006) Why molecules move along a temperature gradient. Proc Natl Acad Sci U S A 103(52):19678–19682. doi:10.1073/pnas.0603873103
Duhr S, Arduini S, Braun D (2004) Thermophoresis of DNA determined by microfluidic fluorescence. Eur Phys J E Soft Matter 15(3):277–286. doi:10.1140/epje/i2004-10073-5
Baaske P, Wienken CJ, Reineck P, Duhr S, Braun D (2010) Optical thermophoresis for quantifying the buffer dependence of aptamer binding. Angew Chem Int Ed Eng 49(12):2238–2241
Ludwig C (1856) Diffusion zwischen ungleich erwärmten Orten gleich zusammengesetzter Lösungen. Sitzungber Bayer Akad Wiss Wien Math, Naturwiss Kl 20
Seidel SA, Wienken CJ, Geissler S, Jerabek-Willemsen M, Duhr S, Reiter A, Trauner D, Braun D, Baaske P (2012) Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding. Angew Chem Int Ed Eng 51(42):10656–10659
Schubert T, Pusch MC, Diermeier S, Benes V, Kremmer E, Imhof A, Langst G (2012) Df31 protein and snoRNAs maintain accessible higher-order structures of chromatin. Mol Cell 48(3):434–444. doi:10.1016/j.molcel.2012.08.021
Zillner K, Jerabek-Willemsen M, Duhr S, Braun D, Langst G, Baaske P (2012) Microscale thermophoresis as a sensitive method to quantify protein: nucleic acid interactions in solution. Methods Mol Biol 815:241–252
Wienken CJ, Baaske P, Rothbauer U, Braun D, Duhr S (2010) Protein-binding assays in biological liquids using microscale thermophoresis. Nat Commun 1:100
Seidel SA, Dijkman PM, Lea WA, van den Bogaart G, Jerabek-Willemsen M, Lazic A, Joseph JS, Srinivasan P, Baaske P, Simeonov A, Katritch I, Melo FA, Ladbury JE, Schreiber G, Watts A, Braun D, Duhr S (2013) Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 59(3):301–315
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Mueller, A.M., Breitsprecher, D., Duhr, S., Baaske, P., Schubert, T., Längst, G. (2017). MicroScale Thermophoresis: A Rapid and Precise Method to Quantify Protein–Nucleic Acid Interactions in Solution. In: Kaufmann, M., Klinger, C., Savelsbergh, A. (eds) Functional Genomics. Methods in Molecular Biology, vol 1654. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7231-9_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7231-9_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7230-2
Online ISBN: 978-1-4939-7231-9
eBook Packages: Springer Protocols