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Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening.

Khong Y Ng1, Kazuya Machida2

  • 1Raymond 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.

Methods in Molecular Biology (Clifton, N.J.)
|January 17, 2017
PubMed
Summary

The rosette assay offers a customizable method for validating SH2 domain interactions. This dot blot technique efficiently assesses binding between SH2 domains and various ligands, aiding in drug screening.

Keywords:
Dot blotGST fusion proteinProtein interaction domainReverse-phase assaySH2 domainTyrosine phosphorylation

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • High-throughput studies and databases enable in silico prediction of protein-protein interactions, specifically SH2 domain interactions.
  • In silico predictions for SH2 domain interactions require experimental validation due to potential unreliability.

Purpose of the Study:

  • To describe a practical protocol for the rosette assay to assess interactions between synthesized tyrosine phosphorylated peptides and a library of GST-tagged SH2 domains.
  • To highlight the broad applicability of the rosette assay for ligand and drug screening beyond SH2-pTyr interactions.

Main Methods:

  • The rosette assay is a dot blot-based, reverse-phase assay for assessing binding between SH2 domains and their ligands.
  • The assay is performed in a 96-well plate, incubating a sample-spotted membrane with labeled SH2 domains.
  • Bound domains are detected and quantified using chemiluminescence or near-infrared fluorescence imaging.

Main Results:

  • The rosette assay is customizable for low- to high-throughput analysis of SH2 domain-ligand interactions.
  • It can analyze interactions with various ligands, including short peptides, purified proteins, and cell lysates.
  • The protocol specifically details assessing interactions between tyrosine phosphorylated peptides and GST-tagged SH2 domains.

Conclusions:

  • The rosette assay provides a reliable experimental validation method for SH2 domain interactions predicted in silico.
  • This versatile technique can be broadly applied to screening different protein interaction domains or antibodies for ligand and drug discovery.