Lab Expectations

The massive amount of DNA in the human body is truly baffling. Stretched end to end, the DNA from a single somatic cell is about 2 meters in length, doing the same for all the DNA in the average human would reach the end of the solar system and back!

Researchers use chromatin immunoprecipitation, or ChIP, to identify and characterize protein-DNA interactions in the context of chromatin. ChIP experiments can use varying input samples, chromatin fragmentation methods, and provide ChIP-qPCR or ChIP-seq readouts.

The activity of genes and their regulatory elements is, in part, governed by their cell type-specific chromatin organization. Nucleosomes, the building blocks of chromatin, are wrapped around a core of histone proteins that are subject to many post-translational modifications that can either promote or silence gene expression.

Antibodies targeted to histone modifications may bind non-specifically to similar, but off-target histone modifications. Conversely, their specific binding can be inhibited by steric hindrance from modifications on neighboring residues. Assays like ELISA, western blot, ChIP, and IF are commonly used to demonstrate antibody specificity and sensitivity, but they cannot clearly predict how an antibody will interact with nearby epitopes. As a result, an alternative approach is needed when trying to validate an antibody to a histone modification target. Continue reading to see how we do it.