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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Kaushansky A, Allen JE, Gordus A et al (2010) Quantifying protein-protein interactions in high throughput using protein domain microarrays. Nat Protoc 5:773–790
Gong W, He K, Covington M et al (2008) The development of protein microarrays and their applications in DNA–protein and protein–protein interaction analyses of Arabidopsis transcription factors. Mol Plant 1:27–41
Ramachandran N, Hainsworth E, Bhullar B et al (2004) Self-assembling protein microarrays. Science 305:86–90
Ramachandran N, Raphael JV, Hainsworth E et al (2008) Next-generation high-density self-assembling functional protein arrays. Nat Methods 5:535–538
Zarate X, Henderson DC, Phillips KC et al (2010) Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts. Proteome Sci 8:32
Kimzey MJ, Zarate X, Galbraith DW et al (2011) Optimizing microarray-based in situ transcription and translation of proteins for matrix-assisted laser desorption ionization mass spectrometry. Anal Biochem 414:282–286
Bourzac KM, Rounseville MP, Zarate X et al (2011) A high-density quantitative nuclease protection microarray platform for high-throughput analysis of gene expression. J Biotechnol 154:68–75
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-62703-782-2_21
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-781-5
Online ISBN: 978-1-62703-782-2
eBook Packages: Springer Protocols