Eliza Small, PhD
Protein and Cell Analysis
Thermo Fisher Scientific
Eliza Small, PhD, is a scientist working on the Invitrogen antibodies portfolio for epigenetics research. She’s leading a collaboration with EpiCypher on a new way to identify highly specific histone antibodies for chromatin immunoprecipitation (ChIP). EpiCypher has a technology called SNAP-ChIP (with “SNAP” meaning “sample normalization and antibody profiling”) that is being used to rigorously validate our histone antibodies. We sat down with her to understand what makes this new technique unique, innovative, and powerful.
SNAP-ChIP is a technology developed by Alex Ruthenburg’s lab at the University of Chicago to normalize ChIP experiments and originally was called ICe-ChIP. It works by using recombinant nucleosomes containing a variety of histone modifications and each nucleosome has a unique barcode associated with the DNA wrapped around it. The panel of recombinant nucleosomes is used as a spike-in to a cell lysate followed by the researcher’s favorite ChIP workflow. After pulldown and DNA elution, qPCR is used to quantitate the pulldown efficiency of the recombinant nucleosomes, which can be used to compare ChIP experiments by using this spike-in as an internal normalization. SNAP-ChIP is also a powerful method for determining how specific your antibody is, as you can learn how much of your antibody is pulling down the modification you are interested in compared to a panel of other histone modifications.
A common method for validating histone modification antibodies is peptide arrays. This method is great for a screen of many modifications and to understand if neighboring modifications influence antibody recognition. Arrays, however, are asking the antibody to recognize a spotted peptide, and a ChIP experiment is asking an antibody to recognize a modification in the context of chromatin. There have been hints in the literature for some time that peptide arrays might not be the best method of determining antibody specificity; and recent published work (Shah, RN et al, 2018), along with our own analysis of our portfolio, has shown there is no correlation between the specificity of peptide arrays
Specificity is crucial to your experiment. In ChIP you are identifying where a modification resides on the genome as well as where it does not. With this information, researchers are gaining an understanding of the role of different modifications in gene expression. If your antibody is pulling down the modification you are interested in in addition to other modifications, the data can be very misleading as you will be misidentifying histone occupancy.
SNAP-ChIP brings a new accuracy to ChIP experiments to ensure an antibody is pulling down what we think it should be. That’s what has interested us in the antibody group at Thermo Fisher Scientific, and why we began working with EpiCypher to test our antibodies. SNAP-ChIP also provides an internal normalization control, which gives researchers much greater confidence in making comparisons between ChIP experiments.
Peptide arrays should absolutely be employed, just not for determining ChIP specificity. The lack of correlation between ChIP specificity and peptide array has revealed that specificity for antibodies is application-dependent, at least in the case of histone modifications.
ChIP is a complicated experiment with many steps. Optimization of each step, including choosing antibody, is essential for success. I highly recommend using SNAP-ChIP in your workflow when using histone antibodies. No matter what target you are interested in studying, look at the data provided for an antibody to understand specificity. You want to see regions of the genome enriched where you expect your target, and regions depleted where you do not expect your target. Additional specificity that you might want is a knockdown, knockout, or ChIP-western, where you perform a western blot after the pulldown step. These are all methods that help give confidence that your antibody is specific to the intended target.
For Research Use Only. Not for use in diagnostic procedures.