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Issue 20, 2016
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Two-channel image analysis method for the screening of OBOC libraries

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Abstract

The split-synthesis approach offers a quick and easy method for producing a large diversity of substances for the discovery of novel protein ligands. Screening of the resulting one-bead-one-compound (OBOC) libraries with fluorescently labelled proteins is not trivial, however, since the resin beads can display significant autofluorescence. Here we present a simple two-channel microscopy image-based screening method for OBOC libraries on TentaGel MB-HMBA resin using tracers labelled with fluorophores. Bead images are taken at short exposure times in the RHO and FITC channels to keep photobleaching of the fluorophores at a minimum. Simple RGB colour vector addition ensures the identification of beads with high fluorescence intensities in the RHO channel. Pre-sorting of the library to exclude highly fluorescent beads is not necessary. The presented method is especially suited for small laboratories without automation equipment. By screening a library with a maximum of 117 649 peptoid hexamers we discovered 18 sequences that bind the human chemokine interleukin-8 (CXCL8) with affinities between 11 μM and 112 μM.

Graphical abstract: Two-channel image analysis method for the screening of OBOC libraries

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Publication details

The article was received on 13 Nov 2015, accepted on 28 Apr 2016 and first published on 29 Apr 2016


Article type: Technical Note
DOI: 10.1039/C5AY02981C
Citation: Anal. Methods, 2016,8, 4142-4152
  • Open access: Creative Commons BY-NC license
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    Two-channel image analysis method for the screening of OBOC libraries

    D. Helmer, K. Brahm, C. Helmer, J. S. Wack, G. Brenner-Weiss and K. Schmitz, Anal. Methods, 2016, 8, 4142
    DOI: 10.1039/C5AY02981C

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