Mapping of domain-mediated protein-protein interaction by SPOT peptide assay

STAR Protoc. 2021 Apr 27;2(2):100503. doi: 10.1016/j.xpro.2021.100503. eCollection 2021 Jun 18.

Abstract

Identification of peptides mediating protein-protein interaction (PPI) is crucial for understanding the function of interlinked proteins in cellular processes and amino acid-associated diseases. Traditional PPI assays are laborious, involving the generation of many truncated proteins. SPOT peptide assay allows high-throughput detection of domains essential for PPI by synthesizing several hundred peptides on a cellulose membrane. Here, we present a rapid SPOT peptide protocol for identifying the binding motifs, which mediate interaction between the chromatin remodeling factors BAF155/BAF170 and the epigenetic factor Kdm6b. For complete details on the use and execution of this protocol, please refer to Narayanan et al. (2015).

Keywords: High Throughput Screening; Protein Biochemistry; Protein expression and purification; Proteomics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • HeLa Cells
  • Humans
  • Jumonji Domain-Containing Histone Demethylases / chemistry
  • Jumonji Domain-Containing Histone Demethylases / metabolism*
  • Peptide Library*
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping*
  • Proteomics*
  • Transcription Factors / chemistry
  • Transcription Factors / metabolism*

Substances

  • DNA-Binding Proteins
  • Peptide Library
  • SMARCC1 protein, human
  • SMARCC2 protein, human
  • Transcription Factors
  • Jumonji Domain-Containing Histone Demethylases
  • KDM6B protein, human