Abstract
Self-assembling protein microarrays have recently emerged as a particularly useful and flexible platform supporting high-throughput analyses of proteins and their interactions. They are produced by printing an array containing a tag-specific antibody. The array is then covered with a solution containing a cell-free expression (linked transcription–translation) system and DNA templates encoding tagged fusion proteins. The proteins synthesized in situ are immobilized by the capture antibody at each array element. An efficient cell-free protein expression system is therefore a critical component in the production of these arrays. Here we describe the methodology for the construction of autofluorescent protein microarrays, by transcription and translation of chimeric proteins containing a C-terminal green fluorescent protein (GFP) tag using different cell-free expression systems, based on either an Escherichia coli S30 extract, a wheat germ extract, or a hybrid system which combines both. The method is followed by a modified version that describes the use of fluorescence amplification as a means for detection of protein interactions. This protocol provides more sensitivity for protein detection on the arrays and allows the choice of different protein tags and/or fluorescent dyes.
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Zárate, X., Galbraith, D.W. (2014). A Cell-Free Expression Platform for Production of Protein Microarrays. In: Alexandrov, K., Johnston, W. (eds) Cell-Free Protein Synthesis. Methods in Molecular Biology, vol 1118. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-782-2_21
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DOI: https://doi.org/10.1007/978-1-62703-782-2_21
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-781-5
Online ISBN: 978-1-62703-782-2
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