Do Chip experiment, antibody is the key!

Chromosome immunoprecipitation (ChIP) is a classic technique for studying the distribution of intracellular proteins on chromosomal DNA. These proteins may be histone subunits, transcription factors, or other regulatory proteins or structural proteins that bind directly or indirectly to DNA.

In general, the ChIP experiment is subdivided into several significant steps: sample preparation, protein-DNA cross-linking, cell lysis and chromatin fragmentation, chromatin immunoprecipitation, purification of DNA, and finally quantitative PCR (qPCR) Or sequencing and other methods to determine or screen out the DNA fragment interacting with the target protein.

For successful ChIP experiments, choosing the appropriate ChIP antibody is one of the most crucial steps. Even the highest quality antibodies that perform very well in classical Western blot validation are not necessarily suitable for ChIP. It is best to consider the use of antibodies that have been validated explicitly in ChIP experiments and validated in each batch(Eg, ChIPAb + ™ Antibody Primer Set). Because antibodies to the ChIP assay recognise proteins in the native chromatin state or possible cross-linked conformations or modified residues of interest, there may be results even for different batches of antibodies of the same article number. If your antibody is not individually quality controlled and has been shown to be useful in ChIP, we recommend that you evaluate one of several possible antibodies and then choose the one that best fits your ChIP experiment.

Before you choose your antibody, you must know the specificity of the antibody, which is especially true when antibodies to post-translationally modified proteins(such as modified histones)are used. You must verify that the antibodies used to detect only those epitopes that contain a specific modification site. There are many ways to assess antibody specificity, including dot blotting, peptide chip or peptide inhibition assays and more. Here, we recommend a reliable method for detecting histone antibody specificity - the AbSurance ™ histone antibody-specific chip (Cat. No. 16-665, 16-668 and 16-667). These chips provide a total of 89 high-quality peptides of histones H2, H3 and H4 on PVDF membranes. The analysis is as simple as Western blot. The result is shown below:

Specific interactions were detected using an antibody to Trimethylated histone H3 lysine 4 (Catalog No. 05-745R) by Merck Millipore. The Insurance ™ chip shows the expected specificity of H3K4me3 peptides (9A and 9B, green boxes) without interaction with other peptides. The primary antibody has a dilution of 1: 2,000. Positive control rabbit IgG is displayed at 12H (blue box).

Non-specific interactions were detected using a supplier E antibody to acetylated histone H3 lysine 56. Insurance ™ chip data showed weak recognition of the target peptide H3K56ac (6G and 6H, green boxes) and strong cross-reactivity with H3K9ac

(1C and 1D, red boxes). The primary antibody has a dilution of 1: 2,000. Positive control rabbit IgG is displayed at 12H (blue box).

From: Merck Life Sciences

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