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Quantitative Analysis of Protein–Protein Interactions by Native Page/Fluorimaging

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Abstract

We have developed a new quantitative native PAGE mobility shift assay, which allows for the measurement of binding affinities for interacting protein pairs, one of which is fluorescently labelled. We have used it to examine recognition of the Simian virus 40 (SV40) large tumour T-antigen (T-ag) nuclear localisation sequence (NLS) by members of the importin (Imp) superfamily of nuclear transport proteins. We demonstrate that the T-ag NLS binds to the Imp α/β heterodimer in NLS-dependent manner, determining that it binds with eight-fold higher affinity (340 nM), when compared to Imp α alone, consistent with autoinhibition of Imp αwhen not complexed with Imp β. The mobility shift assay is able to detect nM binding affinities, making it a sensitive and useful tool to analyse protein–protein interactions in solution.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Nuclear Signalling Laboratory, Monash University, Clayton, VIC, 3800, Australia

    Kylie M. Wagstaff, Manisha M. Dias, Gualtiero Alvisi & David A. Jans

  2. ARC Centre of Excellence for Biotechnology and Development, Canberra, Australia

    David A. Jans

Authors
  1. Kylie M. Wagstaff
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  2. Manisha M. Dias
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  3. Gualtiero Alvisi
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  4. David A. Jans
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Correspondence to David A. Jans.

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Wagstaff, K.M., Dias, M.M., Alvisi, G. et al. Quantitative Analysis of Protein–Protein Interactions by Native Page/Fluorimaging. J Fluoresc 15, 469–473 (2005). https://doi.org/10.1007/s10895-005-2819-5

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  • DOI: https://doi.org/10.1007/s10895-005-2819-5

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