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Protein-binding assays in biological liquids using microscale thermophoresis

Author

Listed:
  • Christoph J. Wienken

    (Ludwig-Maximilians-Universität München, Amalienstrasse 54, Munich 80799, Germany.)

  • Philipp Baaske

    (Ludwig-Maximilians-Universität München, Amalienstrasse 54, Munich 80799, Germany.
    NanoTemper Technologies GmbH, Amalienstrasse 54, Munich 80799, Germany.)

  • Ulrich Rothbauer

    (Ludwig-Maximilians-Universität München, Grosshaderner Strasse 2, Martinsried 82152, Germany.)

  • Dieter Braun

    (Ludwig-Maximilians-Universität München, Amalienstrasse 54, Munich 80799, Germany.)

  • Stefan Duhr

    (Ludwig-Maximilians-Universität München, Amalienstrasse 54, Munich 80799, Germany.
    NanoTemper Technologies GmbH, Amalienstrasse 54, Munich 80799, Germany.)

Abstract

Protein interactions inside the human body are expected to differ from the situation in vitro. This is crucial when investigating protein functions or developing new drugs. In this study, we present a sample-efficient, free-solution method, termed microscale thermophoresis, that is capable of analysing interactions of proteins or small molecules in biological liquids such as blood serum or cell lysate. The technique is based on the thermophoresis of molecules, which provides information about molecule size, charge and hydration shell. We validated the method using immunologically relevant systems including human interferon gamma and the interaction of calmodulin with calcium. The affinity of the small-molecule inhibitor quercetin to its kinase PKA was determined in buffer and human serum, revealing a 400-fold reduced affinity in serum. This information about the influence of the biological matrix may allow to make more reliable conclusions on protein functionality, and may facilitate more efficient drug development.

Suggested Citation

  • Christoph J. Wienken & Philipp Baaske & Ulrich Rothbauer & Dieter Braun & Stefan Duhr, 2010. "Protein-binding assays in biological liquids using microscale thermophoresis," Nature Communications, Nature, vol. 1(1), pages 1-7, December.
    • Handle: RePEc:nat:natcom:v:1:y:2010:i:1:d:10.1038_ncomms1093
    DOI: 10.1038/ncomms1093
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