title: Readout of protein microarrays using intrinsic time resolved UV fluorescence for label-free detection.
abstract: Detecting protein-protein interactions other than those of antibody-antigen pairs still represents a demanding and tedious task. In the present work, a novel method as an alternative to current molecular biology-based detection procedures is established. It solely relies on the change of fluorescence decay times of the protein's intrinsic fluorophores tryptophan and tyrosine due to protein-protein interaction. Unlike previously utilized related methods, no labelling of the binding partners is required. This opens the possibility to detect proteins and their natural interactions without perturbation due to chemical alteration. The technique uses immobilization of one of the protein partners onto solid supports, which allows performance of protein binding studies in the microarray format. Fluorescence lifetime experiments of proteins in their different binding states have been applied to protease/protease-substrate pairs, as well as to the tubulin/kinesin system. Different binding behavior of proteins in solution towards protein partners immobilized on protein microarrays was detected with regard to binding specificity and protein amount. This label-free method for analyzing protein microarrays offers broad applicability ranging from principal investigations of protein interactions to applications in molecular biology and medicine.
authors.affiliation: Institut für Molekulare Biotechnologie, Jena, Germany.
authors.forename: Hans-Martin
authors.initials: HM
authors.lastname: Striebel
authors.forename: Peter
authors.initials: P
authors.lastname: Schellenberg
authors.forename: Paulius
authors.initials: P
authors.lastname: Grigaravicius
authors.forename: Karl Otto
authors.initials: KO
authors.lastname: Greulich
mesh_terms: D005453:Fluorescence; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: D011506:Proteins; D014443:Tyrosine; D014364:Tryptophan