Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.DOI: http://dx.doi.org/10.7554/eLife.24903.001

Antibodies have been indispensable tools in molecular biology, biochemistry and medical research. However, a number of issues surrounding validation, specificity and batch variation of commercially available antibodies have prompted research groups to develop novel non-antibody binding reagents. The ability to select highly specific monoclonal non-antibody binding proteins without the need for animals, the ease of production and the ability to site-directly label has enabled a wide variety of applications to be tested, including imaging. In this review, we discuss the success of a number of non-antibody reagents in imaging applications, including the recently reported Affimer.

Cancer is frequently characterised by dysregulation of the cellular signalling processes that govern proliferation, survival and attachment. Understanding such dysregulation continues to present a challenge given the importance of protein-protein interactions in intracellular processes. Exploring this protein-protein interactome requires novel tools capable of discriminating between highly homologous proteins, individual domains and post-translational modifications. This review examines the potential of scaffold-based binding proteins to fulfil these requirements. It also explores protein-protein interactions in the context of intracellular signalling pathways and cancer, and demonstrates the uses of scaffold proteins as functional moderators, biosensors and imaging reagents. This review also highlights the timeliness and potential to develop international consortia to develop and validate highly specific "proteome" scaffold-based binding protein reagents with the ultimate aim of developing screening tools for studying the interactome.

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