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Application of isothermal titration calorimetry and column chromatography for identification of biomolecular targets

Nature Protocols volume 6, pages 158–165 (2011)Cite this article

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Abstract

This protocol describes a method for identifying unknown target proteins from a mixture of biomolecules for a given drug or a lead compound. This method is based on a combination of chromatography and isothermal titration calorimetry (ITC) where ITC is used as a tracking tool. The first step involves the use of ITC to confirm the binding of ligand to a component in the biomolecular mixture. Subsequently, the biomolecular mixture is fractionated by chromatography, and the binding of the ligand with individual fractions (or subfractions) is verified by ITC. The iteration of chromatographic purification on the fractions combined with ITC results in identifying the target protein. This method is useful when the target protein or ligand is unknown and/or not amenable to labeling, chemical modification or immobilization. This protocol has been successfully used by our team and by others to identify both low-abundance and highly abundant target proteins present in biomolecular mixtures. With this protocol, it takes approximately 3–5 d to identify the target protein from a mixture.

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Figure 1: Flowchart for fishing target proteins from a mixture of biomolecules using isothermal titration calorimetry (ITC) and column chromatography.
Figure 2: Isothermal titration calorimetry (ITC) experimental setup.
Figure 3: Identification and isolation of the target protein for suramin using the isothermal titration calorimetric (ITC) tracking protocol.

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Acknowledgements

This work was supported by a Biomedical Research Council (BMRC) grant (R154000273305) from Singapore's Agency for Science Technology and Research (A*STAR), Singapore.

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Authors and Affiliations

  1. Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore

    Xingding Zhou, R Manjunatha Kini & J Sivaraman

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  1. Xingding Zhou
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Contributions

X.Z. designed and conducted the experiments and wrote the paper. R.M.K. and J.S. designed the experiments and wrote the paper.

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Correspondence to J Sivaraman.

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The authors declare no competing financial interests.

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Zhou, X., Kini, R. & Sivaraman, J. Application of isothermal titration calorimetry and column chromatography for identification of biomolecular targets. Nat Protoc 6, 158–165 (2011). https://doi.org/10.1038/nprot.2010.187

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