title: Evaluation of a sensitive detection method for peptide arrays prepared by SPOT synthesis.
abstract: The growing range of applications for peptide arrays prepared by SPOT synthesis confirms that they are a powerful proteomics technique to study numerous aspects of molecular interaction events. The most frequent application for peptide arrays prepared by SPOT synthesis is the identification of linear epitopes that are recognized by antibodies. In the conventional format using secondary antibodies for detection unspecific binding and high background have been observed. This leads to difficulties in evaluation of developed membranes. Especially for application with combinatorial libraries false positive results are to be avoided. To circumvent this issue, we directly labeled compounds of interest with biotin and detected binding by incubation with streptavidin-horseradish peroxidase via chemiluminescence. Optimization of method conditions led to a very sensitive detection technique with no or low number of unspecific spots, which is superior to conventional detection with secondary antibodies. As one consequence, evaluation of competitive assays got more reliable.
authors.affiliation: Department of Biotechnology, Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: A
authors.initials: A
authors.lastname: Dürauer
authors.forename: E
authors.initials: E
authors.lastname: Kopecky
authors.forename: E
authors.initials: E
authors.lastname: Berger
authors.forename: M
authors.initials: M
authors.lastname: Seifert
authors.forename: R
authors.initials: R
authors.lastname: Hahn
authors.forename: A
authors.initials: A
authors.lastname: Jungbauer
mesh_terms: D018604:Epitope Mapping / Q000379:methods; D007202:Indicators and Reagents; D008024:Ligands; D010455:Peptides / Q000032:analysis*; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D012965:Sodium Chloride
chemical_list: D007202:Indicators and Reagents; D008024:Ligands; D010455:Peptides; D012965:Sodium Chloride