The best way to confirm an protein-protein interaction? - Between a protein and an intergrator (Oct/18/2007 )
We are studying a function of a protein (X), which is found in an economically important crop plant. We have isolated and characterized the proteins interaction partners by using a high quality yeast two hybrid prey library, produced from the crop plant that we are working with.
Our question is now: which is the best method to use for studying protein-protein interactions between the protein (X) and a newly isolated interator (Y)? The method should preferably rule out that the interaction is brigded by another protein (Z). Is co-immunoprecipitation the way to go or are there better methods? Which one?
Thanks.
Our question is now: which is the best method to use for studying protein-protein interactions between the protein (X) and a newly isolated interator (Y)? The method should preferably rule out that the interaction is brigded by another protein (Z). Is co-immunoprecipitation the way to go or are there better methods? Which one?
Thanks.
The best way to determine if protein X and protein Y are directly interacting (ie: no bridging protein) is to do a GST pull-down. Essentially you put both proteins in a test tube and see if they interact. You need one protein expressed with a GST tag in either bacteria or baculovirus infected insect cells. You can actually do this with other tags but GST is clean and easy. The other protein you produce in an in vitro transcribe/translate reaction (TNT) and label with 35S-Met. Add some TNT product to the GST-bead bound protein and determine if there was any binding with an autoradiograph. The primary critisism of a GST pulldown is that it's all in vitro and very artificial. You should certainly try to back up any observed binding from the pull down with a CO-IP which is often considered the gold standard of interaction studies. This indicates that the interaction occurs in vivo as well. If you don't see any binding in the pulldown you may have to do CO-IP to determine if it doesn't occur or needs a mediator protein.
Thank you so much for the help! 
Our question is now: which is the best method to use for studying protein-protein interactions between the protein (X) and a newly isolated interator (Y)? The method should preferably rule out that the interaction is brigded by another protein (Z). Is co-immunoprecipitation the way to go or are there better methods? Which one?
Thanks.
you can perform far western blots, co-immuoprecipitations or pull-down assays if there are tagged (not restricted to GST) proteins
I think that you can also express both proteins by the in vitro TNT systems, both as tagged proteins, e.g. 1 with HA and the other with Myc (provided that you don't have the antibodies for them). Then you can do IP with anti-HA and then detect with anti-Myc, and/or vice versa. That way you don't need radioactive. Or else you could do a GST-X pull-down a His-Y.