Chapter 8 Detection of Protein Cage Assembly with Bisarsenic Fluorescent Probes Thomas A. Cornell and Brendan P. Orner Abstract We describe a method for the detection of specific protein-protein interactions in protein cages through the exploitation of designed binding sites for bisarsenic fluorescent probes. These sites are engineered to be protein-protein interface specific. We have adapted this method to ferritins; however, it could conceivably be applied to other protein cages. It is thought that this technique could be utilized in the thermodynamic and kinetic characterization of cage assembly mechanisms and in the high-throughput screening of protein cage libraries for the discovery of proteins with new assembly properties or of optimized conditions for assembly. Key words FlAsH-EDT 2 , Protein-protein interactions, Fluorescence detection, Oligomerization state, Ferritin 1 Introduction Nanocage proteins play key roles across many biological systems. They perform wide-ranging functions, such as metabolite synthesis [1], protein-folding assistance [2], viral genome protection and delivery [3], and ion and metabolite storage and sequestration [4], which are dependent on, and a consequence of, their large, complex, and often self-assembly controlled, structures [5]. Many of the techniques employed to study the formation of these unique architectures are often indirect and provide only low structural and temporal resolution. Because of their unique hollow nanostructure, protein cages are attractive for a wide range of nonnatural applications. For example, they have been employed as vehicles capable of controlled drug deliv- ery and as size-constrained synthetic reaction vessels for mineralized materials [6, 7]. To develop such systems with greater utility, proteins with properties tailored to each specific application are required. However, the rational design of protein cages with bespoke properties faces the dual challenges of engineering both protein-folding and Brendan P. Orner (ed.), Protein Cages: Methods and Protocols, Methods in Molecular Biology, vol. 1252, DOI 10.1007/978-1-4939-2131-7_8, © Springer Science+Business Media New York 2015 79