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Chromatin IP Antibody Validation

Rigorous Testing and Validation to Create Antibodies that Work in ChIP

Chromatin immunoprecipitation (chromatin IP or ChIP) is an extremely challenging technique, and only antibodies of the highest quality will do. Active Motif specializes in manufacturing high-quality antibodies to histones, histone modifications, transcription factors and chromatin proteins, and in validating these antibodies for use in ChIP.

For more information on our overall antibody development and validation process, visit our page on Antibody Development.

To see all of our primary antibodies, please go to the Antibodies page.

The Importance of Chromatin IP

Chromatin immunoprecipitation (Chromatin IP, ChIP) is a widely used technique in many areas of research, including the study of transcriptional regulation, chromosome biology, epigenetics and cancer (to name a few). Because any process that deals with DNA should include the study of DNA in its native chromatin context, ChIP could be applied to the study of almost all processes that involve DNA and chromatin.

The Utility of Chromatin IP

Chromatin IP enables the detection of a chromatin-associated protein or histone modification at specific genomic loci or, using genome-wide approaches, the distribution of the protein or modification across the genome. Re-ChIP enables one to study if two proteins or modifications are present at the same region of the genome at the same time.

Chromatin IP Requires High Quality Antibodies

For an antibody to work in ChIP, it must meet several requirements. It must have high specificity for its target. In the case of histone modification antibodies, they must recognize only the desired modification (e.g. trimethyl vs. dimethyl or monomethyl) and only at the desired amino acid. It must also recognize the target in the context of chromatin, usually formaldehyde-fixed chromatin (unless performing native ChIP on unfixed chromatin). It also usually requires that the antibody be of fairly high titre, so that only a few µl or µg of antibody is required per ChIP.

Chromatin IP Antibodies: Polyclonal vs Monoclonal

Monoclonals have the advantage of providing consistency over time, avoiding the lot-to-lot variability that is sometimes observed with polyclonals. There are some very good monoclonals that work in ChIP but it has been challenging to develop monoclonals that work as well or better than polyclonals to the same target. To see all of our monoclonal antibodies validated for use in chromatin immunoprecipitation, please click here.

Chromatin IP Antibody Validation Process

Active Motif has high standards for the antibodies we manufacture. From target selection and immunogen design to the initial screening by dot blot and/or Western blot, we apply the same stringent guidelines that you would if you were making an antibody in your lab. Because ChIP is such a demanding technique, our ChIP validated antibodies have to meet an even higher standard. For our in house ChIP validated polyclonals, we inject many rabbits because we expect most of the antibodies produced will not meet our demanding criteria.

Finding the proper model system for ChIP validation can be tricky, requiring the right combination of cell type, gene target and even cell growth or stimulation. We have used both endpoint and real time quantitative PCR for ChIP validation, but have now gone exclusively to qPCR, as it allows us to measure the performance of specific antibody lots or formulations and compare them to each other for optimization.

Chromatin IP Validated Antibodies

Active Motif offers over 100 ChIP validated antibodies to important chromatin proteins, transcription factors, histones and histone modifications. Below are links to those products. For a complete listing of all the ChIP validated antibodies we offer, visit our page on ChIP-validated antibodies.