Introduction

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 a qualitative or quantitative label-free complex mixture comparison for Co-IP samples. These methods use label-free quantification of proteomics datasets to compare an IP of interest to a control IP.

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.

Label-Free Experiment

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 a comparison of the peptide peak intensities for each protein between samples to provide an overview of the relative amount of each protein present. This allows background proteins present in control to be discounted, highlighting interesting candidate proteins for further study.

This method is cheap ($200 per sample) and simple. No labeling 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.