By Ryan Hamnett, PhD
Chromatin immunoprecipitation (ChIP) is a technique that uses antibodies to isolate specific proteins and their target DNA binding sequences from a complex biological sample. Controls are essential for confirming the accuracy, reliability and sensitivity of ChIPs, and can assist with troubleshooting in case of spurious results.
| Control | Type of control | Description | Purpose | Notes |
|---|---|---|---|---|
| Bead only control | Negative | Run the ChIP as normal but in the absence of any antibody | Identifies non-specific binding to the beads alone | May not be necessary if performing an isotype control, which will also pick up non-specific binding to beads, but can help with troubleshooting |
| Isotype control | Negative | Run the ChIP as normal but replacing the antibody with an isotype control (mock) antibody | Identifies non-specific binding to the antibody (e.g. to the Fc region) due to its isotype | |
| Knockdown or knockout control | Negative | Run the full ChIP on a sample known not to express the target protein due to knockout or knockdown | Confirms the specificity of the antibody | If this is possible it should be included, but it will often not be possible because knockouts of transcription factors or histone proteins are not viable. Other negative controls are likely sufficient |
| Negative locus control | Negative | Perform qPCR on a genomic region where the protein is known not to bind | Confirms specificity of ChIP experiment – no products should be produced | |
| No template control for PCR | Negative | Perform qPCR without any DNA input | Identifies products being amplified from contamination | |
| Input control | Positive | Retain 1-2% of the starting chromatin (after fragmentation but before antibody or bead addition) to run in downstream analysis | Allows normalization to determine the extent of enrichment of a given DNA sequence in the ChIP sample compared to the starting material | Typically run for every ChIP |
| Positive antibody control | Positive | Perform ChIP using an antibody against an abundant, well-characterized protein | Confirms that ChIP protocol is working as expected | Rpb1 (the largest subunit of RNA polymerase II) and histone H3 are common positive controls |
| Positive locus control | Positive | Perform qPCR on a genomic region where the protein is known to bind | Confirms that ChIP has worked as expected |
Table 1:Key controls to run alongside a ChIP experiment.