Assignment Task: Consider the following hypothetical problem:
A researcher approaches you for help with an experimental problem. She has successfully inserted into C. elegans an epitope-tagged version of Gamma-enolase, with two copies of the HA peptide sequence inserted at the N-terminus. This recombinant protein has replaced the original chromosomal copy, so she is confident it is being expressed at normal amounts and in the correct intracellular location. She is now aiming to characterise the Gamma-enolase complex. She has used an anti-HA peptide antibody and protein-A beads to purify the tagged recombinant protein along with a functional Gamma-enolase complex. Initial SDS-PAGE and Coomassie staining of the purified complex showed the presence of a series of distinct protein gel bands.
1. Give two examples of other classes of molecules that need to be considered because they can interact with proteins inside the cells to form a functional complex
2. Explain how reverse interaction identifications can be used in pull down experiments to confirm protein-protein interactions
3. Explain how combining a chemical isotopic labelling quantitative proteomics approach such as iTRAQ with immunoprecipitation pull down experiments can be used to distinguish interacting proteins from background contaminants.