One of the most common types of experiment submitted for analysis in the Proteomics Core is the Co-IP experiment. An antibody against endogenous or tagged protein is used to pull down a protein of interest. The goal of the experiment is to see what comes down with that protein, and is a potential interactor. Often Co-IPs will be compared across different treatments or conditions.
Performing a Co-IP experiment with the core
We offer two basic methods for Co-IP experiments:
- Qualitative or quantitative label-free complex mixture comparison.
- SILAC labelled quantitative complex mixture comparison.
These methods use label-free or labelled quantification of proteomics datasets to compare an IP of interest to a control IP, and have their own benefits and weaknesses. We recommend beginning with the much cheaper, simpler label-free method. You may need to proceed to the SILAC method in certain circumstances.
Historically researchers run a control and sample IP on a gel, and look for distinct bands after silver or Coomassie staining. We strongly recommend against this approach. By analysing the entire IP as a complex mixture we can usually identify far more differences than can be seen by eye on a stained gel. The mass-spectrometer is more sensitive than staining – particularly when analyzing a complex mixture, where sample losses are less problematic than in the analysis of weak pure samples or gel bands.
For the label-free experiment you submit a control and treatment IPs as complex mixtures, by running the whole of each sample just 5-10mm into an SDS-PAGE gel, staining with Coomassie, and cutting out the stained region. Follow our complex mixture instructions.
We run the samples on the mass spectrometer and then analyze them. Identification of proteins is performed across all samples, to give consistent resolution of protein grouping / isoform ambiguity etc. We use normalized spectral index quantification to provide an overview of how much of each protein is present in each sample. This allows background proteins present in control to be discounted, highlighting interesting candidate proteins for further study.
This method is cheap ($180 per sample), and simple. No labelling is required. It works well for experiments where you are looking for presence/absence or large fold changes in protein abundance between samples. It cannot provide accurate quantitative ratios for weaker proteins in a sample, which are observed with only a few peptides.
We have delivered useful results to a large number of researchers with this workflow. It is far cheaper, and outperforms, cutting out unique bands from a resolved gel, and submitting them for individual protein ID.
While the label-free quantitation method we use is great for finding large differences and presence/absence between samples it cannot provide very accurate quantitative ratios for the proteins throughout a sample. SILAC labeling offers a method to obtain better quantitative data. We offer a SILAC analysis service, discussed at length on another page.
A method called Isotopic Differentiation of Interactions as Random or Targeted (I-DIRT) uses SILAC labeling to offer enhanced discrimination between background and targeted interactions over our label-free method. It is described in a manuscript by Tackett et. al..
If you suspect your experiment may benefit from a SILAC approach you should speak to us before starting work. Careful preliminary work is necessary to ensure the success of a SILAC experiment.