title: Bead-Based Peptide Arrays for Profiling the Specificity of Modification State-Specific Antibodies.
abstract: Modification state-specific antibodies are powerful tools for investigating posttranslational modifications in proteins. The majority of these antibodies have been generated against peptide-antigen conjugates. They are useful in a plethora of methods, such as Western blotting, immunohistochemistry, sandwich immunoassay, immunoprecipitation, and immunoprecipitation coupled with mass spectrometry. Phosphorylation, acetylation, methylation, sulfation, nitrosylation, ubiquitination, and sumoylation are some of the modifications that can be studied using such antibodies. However, investigating the on- and off-target binding of antibodies is crucial to the interpretation of experimental data. Peptide arrays are excellent tools for such in-depth studies of off-target and on-target binding of antibodies. Dozens or even hundreds of modified peptides can be integrated into a single experimental setup to analyze the antibody's binding behavior. Here, we propose three different protocols for peptide bead array generation and describe their suitability for such types of assay.
authors.affiliation: Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstr. 55, 72770, Reutlingen, Germany.
authors.forename: Angela
authors.initials: A
authors.lastname: Filomena
authors.affiliation: Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstr. 55, 72770, Reutlingen, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Beiter
authors.affiliation: Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstr. 55, 72770, Reutlingen, Germany.
authors.forename: Markus F
authors.initials: MF
authors.lastname: Templin
authors.affiliation: Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstr. 55, 72770, Reutlingen, Germany.
authors.forename: Thomas O
authors.initials: TO
authors.lastname: Joos
authors.affiliation: Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstr. 55, 72770, Reutlingen, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Schneiderhan-Marra
authors.affiliation: Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstr. 55, 72770, Reutlingen, Germany. Oliver.Poetz@nmi.de.
authors.forename: Oliver
authors.initials: O
authors.lastname: Poetz
mesh_terms: D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D040081:Protein Array Analysis / Q000379:methods*
chemical_list: D000906:Antibodies; D000939:Epitopes; D010455:Peptides
keywords: Acetylation; Antibody characterization; Methylation; Peptide bead array; Phosphorylation; Posttranslational modification