title: Microarray-based kinetic colorimetric detection for quantitative multiplex protein phosphorylation analysis.
abstract: Commonly used colorimetric detection applied to protein microarrays with enzymatic signal amplification leads to non-linear signal production upon increase in analyte concentration, thereby considerably limiting the range and accuracy of quantitative readout interpretation. To extend the detection range, we developed a kinetic colorimetric detection protocol for the analysis of ELISA microarrays designed to measure multiple phosphorylated proteins using the platforms ArrayTube™ and ArrayStrip™. With our novel quantification approach, microarrays were calibrated over a broad concentration range spanning four orders of magnitude of analyte concentration with picomolar threshold. We used this design for the simultaneous quantitative measurement of 15 phosphorylated proteins on a single chip.
authors.affiliation: Department of Biology, Institut für Pharmazie und molekulare Biotechnologie, Ruperto-Carola University of Heidelberg, Heidelberg, Germany.
authors.forename: Pavlo
authors.initials: P
authors.lastname: Holenya
authors.forename: Igor
authors.initials: I
authors.lastname: Kitanovic
authors.forename: Florian
authors.initials: F
authors.lastname: Heigwer
authors.forename: Stefan
authors.initials: S
authors.lastname: Wölfl
mesh_terms: D000818:Animals; D003124:Colorimetry / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000378:metabolism; D051379:Mice; D016011:Normal Distribution; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*; D051381:Rats; D012044:Regression Analysis
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins