I want to find new interacting partners for my protein of interest(POI), but I do not want to follow the more conventional and time taking methods like the yeast two hybrid system which also may give false positives and negatives.Taking a Co-IP approach is feasible,but it is established that my POI heterodimerizes with another protein-X most of the time and almost all the phases of cell cycle, so I doubt that an extremely transient interacting partner may be missing from this screen approach. Can anyone suggest probable options which may help me screen for these new partners for POI?
Also, does anyone know of any array based methods for interacting partner screening?
Then I can suggest you following two experiments -
1. Clone your POI with GST tag and confirm its expression and also the tag if everything is in order. Prepare the protein lysate from the cells transfected with plasmid with your POI (GST-tagged). Add antibody against protein X to the lysate, incubate, pass through sepharose beads (this step is to remove protein X). Use the flow through for GST pull down experiment and eluted proteins can be determined by mass spec.
2. For this step you need an antibody against your POI
Follow the same steps until passing through the beads to remove protein X and then follow Co-IP protocol for flow through using antibody against your POI followed by mass spec.
In both the methods you increase the expression of your POI by overexpression. In case you just want to do co-IP with endogenous levels of your POI, then you can simply prepare protein lysate of whichever cell system you are interested, follow the same steps to remove or reduce the levels of protein x (But I am afraid, in this case, your POI might as well bind to the beads as its a strong binding partner of protein x). Flow through can be used for co-IP using antibody against your POI.
What evidence do you have that indicates that your POI is binding to other proteins, besides protein-X?
I think you need to elaborate your question a bit. I am not sure if you already know some putative interaction partner which you want to confirm with the Co-IP or you want to know all the possible interaction partners of your POI?
Anyhow, I can suggest that you can perform GST pull down.
To get rid of protein X (which you say interacts and masks the interaction of other proteins), while performing Co-IP, during pre-clearing step, you can add antibody against Protein X, pass it through the beads and use the eluted protein sample for further processing. This way, you will exclude, or at least remove most of the protein X and its undesired effects will be taken care of.
I hope this is helpful! All the best for your experiment.
Elaboration-My POI has been reported to interact with X under most (if not all) conditions to carry on its functions.Only in limited literature (cell line specific studies) it has been reported that POI carries on its function without the aid of X.I want to find whether POI may carry out the function independently (via homo-dimerization,even though literature survey reveals that it has got a weak homo-dimerization ability) or in association with another protein (which I intend to find) which may compensate X for my POI.
How are you planning to identify new interaction partners after Co-IP? by mass spec?
Then I can suggest you following two experiments -
1. Clone your POI with GST tag and confirm its expression and also the tag if everything is in order. Prepare the protein lysate from the cells transfected with plasmid with your POI (GST-tagged). Add antibody against protein X to the lysate, incubate, pass through sepharose beads (this step is to remove protein X). Use the flow through for GST pull down experiment and eluted proteins can be determined by mass spec.
2. For this step you need an antibody against your POI
Follow the same steps until passing through the beads to remove protein X and then follow Co-IP protocol for flow through using antibody against your POI followed by mass spec.
In both the methods you increase the expression of your POI by overexpression. In case you just want to do co-IP with endogenous levels of your POI, then you can simply prepare protein lysate of whichever cell system you are interested, follow the same steps to remove or reduce the levels of protein x (But I am afraid, in this case, your POI might as well bind to the beads as its a strong binding partner of protein x). Flow through can be used for co-IP using antibody against your POI.
Arnab, I would first of all use an analytical approach on both normal cells and on the cell lines which show a slightly different behaviour: as Ashraf suggests, you need a good Ab against your protein of interest, then the critical step should be the use of a reducible bifunctional aldehyde to stabilize the transient interactors. Then you might have a chance to co-IP your protein with all the interactors. In this case, I would then perform a classical 2D- electrophoresis followed by MS to identify the interactors in the different cell types (and possibly in different conditions).
Thanks a lot for all the valuable suggestions.I will get back to you people if I encounter any problems in the future.
-Cheers
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