◆What’s the difference between Co-IP and GST-pull down?
Co-immunoprecipitation experiment is processed under non-denaturing conditions which can preserve natural activity of interactions between proteins to the largest content. Therefore, Co-IP can well reflect the real protein interaction in situ. However, only with Co-IP we can’t decide whether the protein interaction is direct or indirect. After co-immunoprecipitation by antibodies of target protein, we get a protein complex which not only contains proteins that directly interact with target protein.
For GST-pull down test, we can change experimental conditions in vitro to eliminate effects of non-relevant proteins. Thus we can easily make sure whether target protein interacts with tested protein or not.◆I experience non-specific binding in my immunoprecipitation experiment.
- Use more stringent washing buffer for washing.
- Add a non-ionic detergent (Tween-20 or Triton X-100) to the washing buffer, in concentrations between 0.01-0.1.
- If the beads are blocked before precipitation, add identical blocker to the washing buffer.
- Increase the number of washing steps or prolong the washing steps.
- Decrease incubation time (beads and sample).
- Decrease the antibody concentration.
- A pre-clearing step is prefered to remove molecules that non-specifically bind to the protein A/protein G or the beads themselves.