High-Throughput assessment of ChIP-grade capability with Luminex xMAP technology

Speaker

Abstract

Histone post-translational modifications (PTMs) play pivotal roles in chromatin dynamics and function, with alterations in the healthy profile associated with diverse human pathologies. The regulation and genomic distribution of PTMs is frequently studied by antibodies, despite the wide suspicion that a significant proportion of these reagents have specificity issues. To address this issue we have developed a high -sensitivity and -throughput approach that leverages the multiplexing ability of xMAP technology. We have created a library of >100 biotinylated PTM-containing designer nucleosomes (dNucs) that can be easily combined with Avidin-coated MagPlex beads and determined the binding profiles of >400 commercial ‘PTM-specific’ antibodies. Our results identify truly capable reagents to a large number of PTM targets, but also provide direct evidence that many antibodies in widespread use are highly cross-reactive. As such the current gold-standard screening approaches (immobilized histone PTM-peptide arrays) have proven poorly predictive of alternate assay platforms. In contrast Luminex assays with PTM-nucleosome substrates can be used to identify antibodies with true Chromatin Immuno- Precipitation (ChIP)-grade potential: this will be integral in creating new reagents to study these disease-relevant targets.


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High-Throughput assessment of ChIP-grade capability with Luminex xMAP technology


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