Chapter title |
Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening
|
---|---|
Chapter number | 26 |
Book title |
SH2 Domains
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6762-9_26 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6760-5, 978-1-4939-6762-9
|
Authors |
Khong Y. Ng, Kazuya Machida |
Editors |
Kazuya Machida, Bernard A. Liu |
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. |
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