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Nat Protoc. 2014 Jan;9(1):90-103. doi: 10.1038/nprot.2013.167. Epub 2013 Dec 12.

Discovery of protein interactions using parallel analysis of translated ORFs (PLATO).

Author information

1
1] Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts, USA. [2] Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [3] Department of Genetics, Harvard University Medical School, Boston, Massachusetts, USA. [4] Department of Medicine, Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts, USA. [5] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. [6].
2
1] Department of Medicine, Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts, USA. [2] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
3
1] Department of Genetics, Harvard University Medical School, Boston, Massachusetts, USA. [2] Department of Medicine, Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts, USA. [3] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
4
1] Department of Genetics, Harvard University Medical School, Boston, Massachusetts, USA. [2] Department of Medicine, Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts, USA. [3] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. [4].

Abstract

Parallel analysis of translated open reading frames (ORFs) (PLATO) can be used for the unbiased discovery of interactions between full-length proteins encoded by a library of 'prey' ORFs and surface-immobilized 'bait' antibodies, polypeptides or small-molecular-weight compounds. PLATO uses ribosome display (RD) to link ORF-derived mRNA molecules to the proteins they encode, and recovered mRNA from affinity enrichment is subjected to analysis using massively parallel DNA sequencing. Compared with alternative in vitro methods, PLATO provides several advantages including library size and cost. A unique advantage of PLATO is that an alternative reverse transcription-quantitative PCR (RT-qPCR) protocol can be used to test binding of specific, individual proteins. To illustrate a typical experimental workflow, we demonstrate PLATO for the identification of the immune target of serum antibodies from patients with inclusion body myositis (IBM). Beginning with an ORFeome library in an RD vector, the protocol can produce samples for deep sequencing or RT-qPCR within 4 d.

PMID:
24336473
PMCID:
PMC4129458
DOI:
10.1038/nprot.2013.167
[Indexed for MEDLINE]
Free PMC Article

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