Quantitative analysis of protein-protein interactions by native page/fluorimaging.
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 alpha/beta heterodimer in NLS-dependent manner, determining that it binds with eight-fold higher affinity (340 nM), when compared to Imp alpha alone, consistent with autoinhibition of Imp alpha when not complexed with Imp beta. 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.
Wagstaff
Kylie M
KM
Department of Biochemistry and Molecular Biology, Nuclear Signalling Laboratory, Monash University, Clayton, VIC 3800, Australia.
Dias
Manisha M
MM
Alvisi
Gualtiero
G
Jans
David A
DA
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000737:chemistry; D028884:Karyopherins / Q000737:chemistry; D049408:Luminescent Agents / Q000032:analysis* / Q000737:chemistry; D019913:Nuclear Localization Signals / Q000737:chemistry; D025941:Protein Interaction Mapping / Q000379:methods*; D012680:Sensitivity and Specificity
D000952:Antigens, Polyomavirus Transforming; D028884:Karyopherins; D049408:Luminescent Agents; D019913:Nuclear Localization Signals; D049452:Green Fluorescent Proteins