A cell-free expression platform for production of protein microarrays

Xristo Zárate, David W Galbraith

Research output: Chapter in Book/Report/Conference proceedingChapter

5 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages297-307
Number of pages11
Volume1118
ISBN (Print)9781627037815
DOIs
StatePublished - 2014

Publication series

NameMethods in Molecular Biology
Volume1118
ISSN (Print)10643745

Keywords

  • Cell-free protein expression
  • Green fluorescent protein
  • Hybrid cell-free protein expression
  • Protein microarrays
  • S30 extract
  • Tyramide signal amplification
  • Wheat germ extract

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

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  • Cite this

    Zárate, X., & Galbraith, D. W. (2014). A cell-free expression platform for production of protein microarrays. In Methods in Molecular Biology (Vol. 1118, pp. 297-307). (Methods in Molecular Biology; Vol. 1118). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-782-2_21