title: A cell-free expression platform for production of protein microarrays.
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.
authors.affiliation: Facultad de Ciencias Quimicas, Universidad Autonoma de Nuevo Leon, UANL, Av. Universidad S/N, Ciudad Universitaria, San Nicolas de los Garza, Nuevo Leon, Mexico.
authors.forename: Xristo
authors.initials: X
authors.lastname: Zárate
authors.forename: David W
authors.initials: DW
authors.lastname: Galbraith
mesh_terms: D002457:Cell Extracts; D002474:Cell-Free System; D004926:Escherichia coli / Q000166:cytology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D014176:Protein Biosynthesis
chemical_list: D002457:Cell Extracts; D049452:Green Fluorescent Proteins