Journal of Chromatography B, 815 (2005) 77–95 Review High-throughput and multiplexed protein array technology: protein–DNA and protein–protein interactions Vehary Sakanyan a,b,∗ a Biotechnologie, Biocatalyse et Bior´ egulation, UMR CNRS 6204, Facult´ e des Sciences et Techniques, Universit´ e de Nantes, 2 rue de la Houssini` ere, F-44322 Nantes Cedex 3, France b ProtNeteomix, Nantes, France Received 2 July 2004; accepted 6 August 2004 Available online 27 September 2004 Abstract Miniaturized protein arrays address protein interactions with various types of molecules in a high-throughput and multiplexed fashion. This review focuses on achievements in the analysis of protein–DNA and protein–protein interactions. The technological feasibility of protein arrays depends on the different factors that enable the arrayed proteins to recognize molecular partners and on the specificity of the interactions involved. Proteome-scale studies of molecular interactions require high-throughput approaches for both the production and purification of functionally active proteins. Various solutions have been proposed to avoid non-specific protein interactions on array supports and to monitor low-abundance molecules. The data accumulated indicate that this emerging technology is perfectly suited to resolve networks of protein interactions involved in complex physiological and pathological phenomena in different organisms and to develop sensitive tools for biomedical applications. © 2004 Elsevier B.V. All rights reserved. Keywords: Microarray technologies; Proteomics; Protein array; Protein interactions; High-throughput screening Contents 1. Introduction ......................................................................................................... 78 2. High-throughput protein production .................................................................................... 78 2.1. DNA cloning .................................................................................................. 78 2.2. Affinity purification and solubility of proteins ..................................................................... 80 2.3. Cell-free protein synthesis ...................................................................................... 81 2.4. Automation of protein production ................................................................................ 82 3. Supports for arraying proteins ......................................................................................... 82 3.1. Oriented immobilization ........................................................................................ 82 3.2. Hydrogel ...................................................................................................... 83 3.3. Nitrocellulose membrane ....................................................................................... 83 3.4. Other supports and micro-formats ............................................................................... 83 4. Detection of signal ................................................................................................... 84 4.1. Protein labeling by cell-free synthesis ............................................................................ 84 4.2. Enhancing signals from microspots .............................................................................. 84 ∗ Tel.: +33 2 51 125620; fax: +33 2 51 125637. E-mail address: vehary.sakanyan@chimbio.univ-nantes.fr. 1570-0232/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2004.08.045