Assessing the in vitro Binding Specificity of Histone Modification Reader Proteins Using Histone Peptide Arrays

• Published in: Bio-protocol Vol. 11; no. 18; p. e4168
• Main Authors: Soo, Mark W, Saltzman, Arneet L
• Format: Journal Article
• Language: English
• Published: United States Bio-Protocol 20-09-2021; Bio-protocol LLC
• Subjects: Biology; Methods; Chromatin; Histone peptide array; Chromodomain; Epigenetics; Histone modifications; Histone tail; Recombinant protein; Affinity purification
• ISSN: 2331-8325; 2331-8325
• DOI: 10.21769/BioProtoc.4168

In the field of chromatin biology, a major goal of understanding the roles of histone post-translational modifications is to identify the proteins and domains that recognize these modifications. Synthetic histone peptides containing one or more modifications are a key tool to probe these interactions in pull-down assays with recombinant proteins or cell lysates. Building on these approaches, the binding specificity of a protein of interest can be screened against many histone peptides in parallel using a peptide array. In this protocol, we describe the expression and purification of a recombinant protein of interest in bacteria, followed by an assay for binding to histone post-translational modifications using a commercially available histone peptide array. The purification uses a versatile dual-tagging and cleavage strategy and equipment commonly available in a molecular biology laboratory. Graphic abstract: Overview of protocol for purifying recombinant protein and hybridizing to a histone peptide array.