Immunoprecipitation and pull-down assays are common techniques for the enrichment of interaction partners of target proteins. Good experiment design and sample preparation are the key to meaningful results in subsequent protein identification by mass spectrometry.
We know that you invest much effort and time to obtain enriched proteins and that these samples are very precious. To maximise the relevance of an experiment's outcome, we have a few recommendations.
- Immobilise the bait or antibody preferably by covalent binding. This dramatically facilitates the purification of target proteins which are present in much smaller amounts. A protocol for conjugation to magnetic beads can be found here. As an example Dynabeads M-270 epoxy are suitable for covalent antibody binding.
- Separate the enriched fraction by SDS-PAGE. If you can visualise target proteins by Coomassie or MS-compatible silver staining, you have enough material for protein identification by mass spectrometry.
- Compare with the complex protein mixture applied to IP/pull-down by SDS-PAGE. Run control samples. Then you can choose relevant proteins for subsequent identification.
Please cut out only single bands of interest (i. e. 1-3 mm in height). Larger gel pieces result in unfavourably low ratio of target analyte to proteolytic background which is especially critical at the commonly encountered low amounts of target analyte. For the same reason we discourage to do direct analyses of IP eluates.