The field of epigenetics and the study of chromatin structure have begun to shed new light on the regulation of gene expression, and how these processes are altered in disease. Chromatin structure is controlled in part by histone post-translational modifications (PTMs), which impact the accessibility of chromatin to transcription factors and other regulatory proteins. Interestingly, distinct histone PTMs denote specific genomic loci, such as promoters, enhancers, and silenced heterochromatin. Thus, scientists have significant interest in mapping these modifications in healthy and diseased tissues, both to learn how changes impact disease development as well as how these marks may be targeted for therapy. Of the many procedures available for chromatin profiling, Chromatin Immunoprecipitation combined with next-generation sequencing (ChIP-seq) is the most commonly used approach for mapping histone PTMs.
There are several prerequisites required in the design of a ChIP experiment, that will help you secure a high level of success. First, you must identify the target proteins you wish to map, whether they are transcription factors or histone modifications. You’ll also want to determine whether you want to proceed with a cross-linked or a native ChIP method, a decision which is impacted by the type of protein or modification you choose to profile. The right ChIP protocol can make the difference between publishable results and data saturated with errors due to background noise and nonspecific signal.
However, the most important criteria influencing the success of your ChIP experiment is the quality of your ChIP Antibody. Selecting the right ChIP antibody requires analysis of several metrics. First and foremost, the antibody needs to show high specificity for its designated target. The identification of a highly specific antibody is crucial for reliable results. Indeed, if your antibody displays cross-reactivity with off-target proteins or marks, then it doesn’t matter how carefully you have planned your protocol or your reasons for profiling this protein. Even if all of the other pieces of your experiment have been highly controlled, the use of a nonspecific ChIP antibody will yield unreliable and/or inaccurate results.
One of the best ways to examine the specificity of an antibody is to test its ability to bind off-target vs. on-target proteins that are relevant in the context of your experiment. For instance, in the case of testing antibodies to H3K4me3, it would be valuable to assess binding of your ChIP antibody to H3K4me0, H3K4me1 and H3K4me2. Using validated antibodies in your experiments can significantly improve the impact of your results.
EpiCypher’s SNAP-ChIPⓇ Certified histone PTM antibodies are the only commercially available antibodies validated against a panel of defined modified recombinant nucleosomes containing both on- and off-target PTMs, all within the context of a ChIP experiment. Validating antibodies with SNAP-ChIP reveals how antibodies behave during a ChIP experiment, including their off-target binding activity and enrichment of on-target PTM (vs Input chromatin). Thus, these methods help reveal the binding efficiency of the antibodies as well as the antibody specificity.
Using EpiCypher SNAP-ChIP certified antibodies can help solve many of the problems inherent to ChIP experiments. They offer low cross-reactivity to help prevent the contamination of your results with nontargeted proteins as well as high IP efficiency. Their antibodies have been extensively tested to ensure a high degree of specificity and low cross-reactivity using spike-in controls to bring you some of the best-validated antibodies for superior ChIP experiments.
The quality of your ChIP data will have profound effects on the accuracy of downstream data interpretation and conclusions. With the rapid pace of change in the field of epigenetics research, it is critical to identify the best reagents to eliminate inefficiency and redundancy. Visit EpiCypher.com today to learn more about best-in-class SNAP-ChIP certified antibodies, and safeguard your ChIP protocols from the pitfalls of low-performance antibodies.