Modified Histone Peptides Linked to Magnetic Beads Reduce Binding Specificity

Abstract

Histone post-translational modifications are small chemical changes to the histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET-based systems. High-throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet, published studies on their use with histones have been noticeably sparse. Here, we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding.

Endoplasmic reticulum sequestration empowers phosphorylation profiling on the yeast surface

“…As an alternative to this approach, we explored magnetic bead selection, which is a well‐established method in selection of yeast‐displayed binding ligands, as well as sorting of large populations in an efficient manner 59 . An earlier study of modified histone peptides linked to magnetic beads presented challenges in binding specificity, but this was likely due to properties of the immobilized short peptides rather than the affinity reagents used for detection 60 . A series of titration experiments led to the optimization of this method in the context of phosphorylated domains.…”

“…59 An earlier study of modified histone peptides linked to magnetic beads presented challenges in binding specificity, but this was likely due to properties of the immobilized short peptides rather than the affinity reagents used for detection. 60 A series of titration experiments led to the optimization of this method in the context of phosphorylated domains. Interestingly, we determined that the amount of protein immobilized did not influence yield on the range tested; however, increasing the amount of magnetic beads used translated into an improvement in recovery.…”