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

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

Bio-protocol (2021-10-26)
Mark W Soo, Arneet L Saltzman
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, pH 7, ≥98%
Sigma-Aldrich
Nalgene® centrifuge tubes, Oak Ridge Style 3114, capacity 50 mL, pack of 2 ea