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Abstract
The increasing involvement of academic institutions and biotech companies in drug discovery calls for cost-effective methods to identify new bioactive molecules. Affinity-based on-bead screening of combinatorial one-bead one-compound libraries combines a split-mix synthesis design with a simple protein binding assay operating directly at the bead matrix. However, one bottleneck for academic scale on-bead screening is the unavailability of a cheap, automated, and robust screening platform that still provides a quantitative signal related to the amount of target protein binding to individual beads for hit bead ranking. Wide-field fluorescence microscopy has long been considered unsuitable due to significant broad spectrum autofluorescence of the library beads in conjunction with low detection sensitivity. Herein, we demonstrate how such a standard microscope equipped with LED-based excitation and a modern CMOS camera can be successfully used for selecting hit beads. We show that the autofluorescence issue can be overcome by an optical image subtraction approach that yields excellent signal-to-noise ratios for the detection of bead-associated target proteins. A polymer capillary attached to a semiautomated bead-picking device allows the operator to efficiently isolate individual hit beads in less than 20 s. The system can be used for ultrafast screening of >200,000 bead-bound compounds in 1.5 h, thereby making high-throughput screening accessible to a wider group within the scientific community.
Original language | English |
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Pages (from-to) | 209-219 |
Number of pages | 12 |
Journal | ACS Combinatorial Science |
Volume | 18 |
Issue number | 5 |
Early online date | 8 Apr 2016 |
DOIs | |
Publication status | Published - 9 May 2016 |
Keywords / Materials (for Non-textual outputs)
- one-bead one-compound libraries
- ultrafast screening
- high-throughput screening
- spectral imaging
- COMBINATORIAL LIBRARIES
- RAPID IDENTIFICATION
- PEPTIDE LIBRARY
- DRUG DISCOVERY
- LIGANDS
- DOMAINS
- HITS
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PRIMES: Protein interaction machines in oncogenic EGF receptor signalling
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