title: Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening.
abstract: With a growing number of high-throughput studies, structural analyses, and availability of protein-protein interaction databases, it is now possible to apply web-based prediction tools to SH2 domain-interactions. However, in silico prediction is not always reliable and requires experimental validation. Rosette assay is a dot blot-based reverse-phase assay developed for the assessment of binding between SH2 domains and their ligands. It is conveniently customizable, allowing for low- to high-throughput analysis of interactions between various numbers of SH2 domains and their ligands, e.g., short peptides, purified proteins, and cell lysates. The binding assay is performed in a 96-well plate (MBA or MWA apparatus) in which a sample spotted membrane is incubated with up to 96 labeled SH2 domains. Bound domains are detected and quantified using a chemiluminescence or near-infrared fluorescence (IR) imaging system. In this chapter, we describe a practical protocol for rosette assay to assess interactions between synthesized tyrosine phosphorylated peptides and a library of GST-tagged SH2 domains. Since the methodology is not confined to assessment of SH2-pTyr interactions, rosette assay can be broadly utilized for ligand and drug screening using different protein interaction domains or antibodies.
authors.affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, 400 Farmington Avenue, Farmington, CT, 06032, USA.
authors.forename: Khong Y
authors.initials: KY
authors.lastname: Ng
authors.affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, 400 Farmington Avenue, Farmington, CT, 06032, USA. machida@uchc.edu.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Machida
mesh_terms: D057166:High-Throughput Screening Assays; D015151:Immunoblotting / Q000295:instrumentation / Q000379:methods*; D010766:Phosphorylation; D019000:Phosphotyrosine; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000295:instrumentation / Q000379:methods*; D011993:Recombinant Fusion Proteins; D018909:src Homology Domains
chemical_list: D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine
keywords: Dot blot; GST fusion protein; Protein interaction domain; Reverse-phase assay; SH2 domain; Tyrosine phosphorylation