Single bead labeling method for combining confocal fluorescence on-bead screening and solution validation of tagged one-bead one-compound libraries.
Screening of one-bead one-compound libraries by incubating beads with fluorescently labeled target protein requires isolation and structure elucidation of a large number of primary hit beads. However, the potency of the identified ligands is only revealed after time consuming and expensive larger scale resynthesis and testing in solution. Often, many of the resynthesized compounds turn out to be weak target binders in solution due to large differences between surface and solution binding affinities. For an industry style high-throughput screening (HTS) process a high false positive rate is detrimental. We have therefore combined single bead and single molecule/single cell techniques into an integrated HTS process in which the picomole amount of substance contained on one isolated hit bead is sufficient for quality control, structure determination, and precise affinity determination to the target protein in solution.
Hintersteiner
Martin
M
University of Edinburgh, School of Biological Sciences, The King's Buildings, CH Waddington Building 3.07, Mayfield Road, Edinburgh EH9 3JR, UK.
Kimmerlin
Thierry
T
Kalthoff
Frank
F
Stoeckli
Markus
M
Garavel
Geraldine
G
Seifert
Jan-Marcus
JM
Meisner
Nicole-Claudia
NC
Uhl
Volker
V
Buehler
Christof
C
Weidemann
Thomas
T
Auer
Manfred
M
D055808:Drug Discovery / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005453:Fluorescence; D008024:Ligands; D008722:Methods; D008863:Microspheres; D015336:Molecular Probe Techniques; D054852:Small Molecule Libraries
D008024:Ligands; D054852:Small Molecule Libraries