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J Proteomics. 2013 Aug 2;88:141-8. doi: 10.1016/j.jprot.2013.02.002. Epub 2013 Feb 20.

Optimised 'on demand' protein arraying from DNA by cell free expression with the 'DNA to Protein Array' (DAPA) technology.

Author information

1
Protein Technology Group, Babraham Bioscience Technologies Ltd., Babraham Research Campus, Cambridge CB22 3AT, UK. ronny.schmidt@babraham.ac.uk

Abstract

We have previously described a protein arraying process based on cell free expression from DNA template arrays (DNA Array to Protein Array, DAPA). Here, we have investigated the influence of different array support coatings (Ni-NTA, Epoxy, 3D-Epoxy and Polyethylene glycol methacrylate (PEGMA)). Their optimal combination yields an increased amount of detected protein and an optimised spot morphology on the resulting protein array compared to the previously published protocol. The specificity of protein capture was improved using a tag-specific capture antibody on a protein repellent surface coating. The conditions for protein expression were optimised to yield the maximum amount of protein or the best detection results using specific monoclonal antibodies or a scaffold binder against the expressed targets. The optimised DAPA system was able to increase by threefold the expression of a representative model protein while conserving recognition by a specific antibody. The amount of expressed protein in DAPA was comparable to those of classically spotted protein arrays. Reaction conditions can be tailored to suit the application of interest.

BIOLOGICAL SIGNIFICANCE:

DAPA represents a cost effective, easy and convenient way of producing protein arrays on demand. The reported work is expected to facilitate the application of DAPA for personalized medicine and screening purposes.

KEYWORDS:

Cell free expression; DAPA; Interaction studies; Protein array; Surface coatings

PMID:
23454659
DOI:
10.1016/j.jprot.2013.02.002
[Indexed for MEDLINE]

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