Question

4. Let’s assume that your experiment in question 3 shows co-localization. However, your advisor suggests that co-localization is not the same things as demonstrating protein-protein interactions between protein X and protein Y. She’s right. Propose a different experiment that can determine if these proteins actually interact as opposed to just co-localize. As always, describe the experiment, the expected data, and the controls you’d use.

Answer 1

A co-immunoprecipitation assay needs to be done with a cell lysate having both the protein X and Y. The protein pull needs to be immunoprecipitated with one protein( antibody against X or Y) and a negative control IgG and then western blot needs to be done. In the western blot, the other protein that was not pulled was checked for and if it is seen to be present in the lane where the cell lysate was pulled with the specific antibody(X or Y) and not in the control IgG lane, then it is concluded that protein X and Y interact.

To further confirm this fact, an in-vitro interaction study needs to be done to check for the protein-protein interaction of X andY.

For this protein X is tagged with 6x-His and purified and protein Y is tagged with GST and purified.

Now these proteins are incubated together in an appropriate buffer and then checked for interaction.

For this two controls are kept,

control1- where only the Protein X is passed through a GSH column.

control2- where only protein Y is passed through a GSH column.

and the experimental setup where the mixture of protein X and Y is passed through a GSH column.

after incubation of the proteins and elution with the addition of excess GSH that help in eluting the bound protein to the column, the eluted proteins are run on a gel and western blots are done.

For the lane in control 1, as protein X does not have a GST tag it does not bind to the column and thus is not observed in the eluted fraction, this shows that protein X does not bind to the column itself.

For the lane in control 2, protein Y does have a GST tag and thus is found in the elution fraction, this ensures that protein Y has a proper affinity to the column.

For the experimental lane, if the proteins interact both the proteins are observed in the elution fraction after western blot is done using antibodies both against Protein X and Y. this proves that protein X and Y interact.

So both the purified system and the co-immunoprecipitation together prove that protein X and Y interact.

Often in cell lysates in the immunoprecipitated complex proteins that have an indirect interaction are also seen to be pulled down, so the purified system is required where only those proteins are present whose interaction has to be monitored and no other protein is there. So both the experiments together prove interaction.