At Abcam we run quite a few ChIP assays and we interact with customers that run even more than us. Lately, we’ve noticed that more researchers are turning to ChIP for the first time as gene regulation studies continue to grow.
So we thought we’d share a couple of quick tips for various aspects of the ChIP method that can be helpful. For a deeper dive into the method, we have both a complete ChIP Tips Guide and a ChIP Troubleshooting Guide that might be useful to you as well.
Validating Antibodies for ChIP Applications
It’s always a good idea to use an antibody that has already been validated in ChIP applications since you’re asking the antibody to do something that is very different from other antibody applications. If you can’t find an antibody that’s already been validated in a ChIP setting, then consider the following steps:
- Perform standard ChIP experiment: Try to find an antibody to your target that has been validated by immunoprecipitation, immunohistochemistry, or immunocytochemistry as they will be more likely to perform well in ChIP applications. Next, perform a standard ChIP experiment. You will need a positive and a negative control locus, i.e. a locus where you know your protein/modification of interest is present and one where you know it is absent or significantly decreased.
- Determine the optimal antibody concentration: Using the correct antibody concentration can significantly improve the signal to background ratio. Titrate the amount of antibody required by performing a ChIP experiment using a range of antibody concentrations. The amount of antibody required per ChIP typically ranges from 1–10 μg of antibody for every 25 μg of chromatin.
- Test different washing conditions: Some antibodies have a low affinity for the target; it is therefore worth testing different washing conditions. The stringency of the last wash can be varied from 250-500 mM salt (usually NaCl or LiCl).
- Verify with a Western Blot: A Western blot can be used to test whether the target has been immunoprecipitated. To test if the antibody pulls down the target of interest you can perform ChIP up to the final wash after the IP and then boil the beads in loading buffer for 10 min. You can then confirm the presence of your protein in the IP by Western blot.
Controls in ChIP Assays
For involved methods like ChIP, good controls are a must. When starting off with ChIP applications, you will want to make sure that you have a good plan for assessing how the experiment is proceeding at the various stages.
Here are a few controls that we’ve used successfully:
- Antibody Controls: As a positive antibody control for ChIP, Histone H3 (trimethyl K4) is a popular positive control to use when chipping active genes. As a negative control, use an antibody that recognizes a non-chromatin epitope such as an anti-GFP antibody.
- Negative Controls: As a negative control, use `beads only` or beads with an isotype matched control immunoglobulin (Ig), this will give you the background of the assay. For example, ab26721, a RNA polymerase II CTD repeat YSPTSPS-ChIP grade antibody, is a mouse monoclonal IgG1. A suitable isotype control would be ab18443, mouse IgG1, Kappa monoclonal isotype control.
- Positive and Negative Control Loci: Primers for a locus where you know your protein or modification of interest is present (positive control locus) and one where it is absent or significantly decreased (negative control locus) should both be tested in RT-PCR. This will tell you whether the observed enrichment is specific. It is important to include these controls as some antibodies result in non-specific enrichment.
- Non-Template Control: A non-template control should always be included in the PCR reaction. This will help you spot any contamination.
- Positive Control Antibody: Antibodies specific for Histone H3 (tri methyl K4) (ab8580) and H3 (acetyl K9) (ab4441), both enriched on actively transcribed regions, are good controls to ensure that each step of your experiment is working.