Journal of Fluorescence, Vol. 15, No. 4, July 2005 (©2005) DOI: 10.1007/s10895-005-2819-5 Quantitative Analysis of Protein–Protein Interactions by Native Page/Fluorimaging Kylie M. Wagstaff, 1 Manisha M. Dias, 1 Gualtiero Alvisi, 1 and David A. Jans 1,2,3 Received February 17, 2005; accepted May 04, 2005 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. KEY WORDS: Protein interactions; polyacrylamide gel electrophoresis; native PAGE mobility shift assay; importins; SV40 T-ag; nuclear localisation signal. INTRODUCTION Quantitative analysis of protein–protein interactions can be complex and time consuming, usually involving the immobilisation of one or other of the interacting pro- tein partners, e.g. in ELISA or biacore based approaches. We have devised a simple technique for quantifying the affinity of solution binding of two proteins under native conditions, one of which is fluorescently labelled, using native polyacrylamide gel electrophoresis (PAGE). As applied here, native PAGE relies on the binding of an unlabelled protein to a fluorescently labelled pro- tein, thus altering the shape/size of the protein, resulting in a shift in the mobility of the fluorophore during na- tive PAGE. Our technique combines this with titration 1 Department of Biochemistry and Molecular Biology, Nuclear Signalling Laboratory, Monash University, Clayton, Australia. 2 ARC Centre of Excellence for Biotechnology and Development, Canberra, Australia. 3 To whom correspondence should be addressed at Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia. E-mail: david. jans@med.monash.edu.au of the unlabelled partner, and analysis of the digitised images obtained to determine the binding affinity of the interaction. As an example of one of the many applica- tions of this assay, we present experiments measuring the binding affinity of proteins from the importin superfamily (Imp) to the nuclear localisation sequence (NLS) of the SV40 large tumour T antigen (T-ag) [1–3]. All transport into and out of a cell nucleus occurs via nuclear pore complexes (NPC) embedded in the nu- clear envelope [3–5]. Active nuclear import is mediated by Imps [6,7] and requires the recognition of an NLS contained within the transport cargo protein [8,9]. In con- ventional nuclear import pathways, the NLS is recognised by the Imp α/β heterodimer through the Imp α subunit, followed by docking at the NPC mediated by the Imp β subunit [7,10]. Translocation into the nucleoplasm is followed by the binding of the monomeric guanine nu- cleotide binding protein Ran, in its GTP bound form, to Imp β to dissociate the complex [9,11]. Imp β facili- tates the Imp α-cargo interaction by binding to Imp α and effecting a conformational change, which releases an au- toinhibitory domain from the NLS binding site of Imp α, thereby greatly increasing the affinity of the interaction 469 1053-0509/05/0700-0469/0 C  2005 Springer Science+Business Media, Inc.