The typical cell is a dynamic entity, always changing, simply because it is "alive". Cellular compartmentalization is also dynamic. Just because two molecules are in separate cellular compartments one moment does not mean they are permanently so. Recall that the nuclear membrane disintegrates when it is time for the cell to divide (mitosis). And there are several more examples of cellular compartments forming and disintegrating depending on the cellular state. Merci et au revoir.

Hi there,

Differential localization of physically interacting proteins is a way to regulate this interaction. Have you followed the dynamics of the protein localization during the different steps of the cell cycle?

And even if you see a clear different localization it doesn't mean there is no interaction at all.

By the way when you mentioned a cytoplasmic localization do you mean the protein is fully excluded from the nucleus (ie. you can spot the nucleus because less stained than the cytoplasm) or do you get a uniform signal within the cell?

Also bear in mind that physical interactions (as measured by Co-IP) need not have any biological validity. Some proteins are simply sticky.

14-3-3s, for example, are basically biological scaffolds, and are thus really good at sticking to other proteins. Chaperonins are similarly good at binding to other stuff.

Interpret Co-IP data with caution, basically.

Hi Shirley,

It is possible that two proteins located in different cellular compartments may interact with each other using CO-IP because all proteins are mixed together once you lyse the cell. A single protein may contain multiple sites that has binding affinity towards other proteins.

I think you can try fractionation to harvest cytosolic proteins and nuclear proteins separately, then combine these samples for CO-IP so you know if its more towards binding affinity or not.

Also, keep in mind that maybe not all protein A can bind to protein B, so you may not able to observe a strong protein-protein interaction signal under microscope.