By Ryan Hamnett, PhD
Co-immunoprecipitation (Co-IP) is a technique that uses antibodies to isolate target protein and their binding partners from a complex biological sample. Controls are essential for confirming the accuracy, reliability and sensitivity of Co-IPs, and can assist with troubleshooting in case of spurious results.
| Control | Type of control | Description | Purpose | Notes |
|---|---|---|---|---|
| Supernatant controls | Negative | Run the supernatant from each main step on the gel and western blot (WB) | Confirms that target protein has successfully been depleted and held (bound by antibody) at each stage | Supernatants from all steps (pre-clearing, IP, washing, elution) can be kept for troubleshooting in case the expected bands are not seen in the experimental lane |
| Bead only control | Negative | Run the co-IP 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 co-IP as normal but replacing the antibody with an isotype control 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 co-IP on a sample known not to express the bait protein due to knockout or knockdown | Confirms the specificity of the antibody | Performing the co-IP on a sample known not to express the protein naturally (e.g. because it is a different tissue type) may also be sufficient |
| Input control | Positive | Run 1-10% of the starting lysate on the gel and WB | 1) Demonstrates IP for the bait was successful: there should be a band in both the IP and input lanes 2) Confirms negative results, e.g. prey successfully blotted for in input but not in IP lanes 3) Indicates co-IP efficiency: comparing strengths of target bands in IP and input lanes 4) Indicates IP specificity: comparing strengths of non-specific bands | Typically run alongside every co-IP. |
| Positive lane control | Positive | Run purified recombinant target protein on the gel and WB | Serves as a positive reference | Running a sample known to express high levels of the protein naturally (e.g. from a different tissue type) may also be sufficient |
| Bidirectional co-IP control | Confirmatory | Once a binding partner has been identified by co-IP, run the co-IP again but reverse the bait and prey | Being able to pull down the same protein complex regardless of which protein is the bait is a useful confirmation of a physiological interaction |
Table 1:Key controls to run alongside an co-IP experiment.