Issue 20, 2016

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

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

Supplementary files

Article information

Article type
Technical Note
Submitted
13 Nov 2015
Accepted
28 Apr 2016
First published
29 Apr 2016
This article is Open Access
Creative Commons BY-NC license

Anal. Methods, 2016,8, 4142-4152

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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