ChIP experiment technique sharing: how to select the antibody of chromatin immunoprecipitation (ChIP) experiment?
In ChIP experiments, the quality of the antibody directly influences the results of the experiment
So how do you select the best antibody in the ChIP experiment and the most critical step in the experiment
Many teachers care about two things in ChIP experiment:
1.In existing antibody ChIP level, how to choose the highest specificity of antibody ChIP, while many manufacturers are able to provide the ChIP level antibody, but in a 2011 Nature article literature report shows that even ChIP level of antibodies, combined with the specificity and only about 70%, how do I choose a high specificity of antibody ChIP level?
The selection of antibody in ChIP experiment is the core element of experiment success.
In the 2011 Nat Struct Mol Biol [1, 2] report, the spot hybridization and ChIP - ChIP test showed that only 73 percent and 78 percent of antibodies had a better specificity (figure 1, figure 2).
The author further by WB test three brand apparent antibody specificity, there is only one brand (Millipore) antibody specificity tests (figure 3), the other two brand fails antibody specificity combined with experiments.
For this kind of circumstance to recommend you a very good method, through the world's largest antibody CiteAb query website (http://www.citeab.com/) in the query, access to literature citations of each ChIP antibody (CiteAb shows Merck's variety of epigenetic antibody global usage ranking first)
2.ChIP level antibody should be selected in the ChIP experiment.
If my experimental species are more specific (fruit flies, insects, plants, etc.) how should I choose ChIP experimental antibodies?
But because of the limited number of antibodies that ChIP verification has done, there are three ways we can think about it when there is no ChIP level antibody:
(1)The IP/IHC/ICC is chosen, and in these experiments, the recognition structure of the antibodies is native, and the ChIP antibody recognition structure is similar;
It is not recommended to try Western blot antibody, because the protein structure identified by WB antibody is denature, which is not suitable for ChIP experiment.
(2)The sequence of proteins is carried out on NCBI. If the protein sequence homology is over 80% with other species or species protein, then the cross-species antibody can be tried.
(3)Label antibodies can be used, but the tabs may interfere with transcription factors and may introduce false positive interactions, so the control needs to be set, including the mock IP control of the untransfected cells;
And convert the label from the n-end to the C.
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