I need to perform a co-immunoprecipitation of an extracellular protein to identify the receptor to which it binds. In order to avoid that the antibody used for the immunoprecipitation recognizes the same regions with which the protein binds the receptor, is there any way to predict the binding sites of the protein to a possible receptor and the binding sites of the antibody to the protein?
the best solution is to clone your protein with a Tag (GST, His, etc), and use the antibody against the tag for IP.
There are several programs you could try for prediction of binding site. In this case you should has the structure of both proteins. Look at PDB databases as: www.rcsb.org
For prediction try http://zdock.umassmed.edu/
Dear Julio.
Is it possible that a tag could alter the fold of my protein and impair protein-receptor interaction?
hi Nicola
between the tag and the protein usually a flexible peptide linker is used, in that way the protein and your tag should be independent domains and the fold of your protein is not altered.
an example of the linker sequence is: GGGGSGGGGS
there are several commercial vectors for this purpose
1. Do you know if a tag could alter the dimerization of the protein of interest?
2. N-terminus of the protein of interest is a possible candidate region for interactions with a putative receptor. Can a N-terminus tag alter the interaction?
1.The dimerization is possible, use a small tag for instance c-myc, his, strep-tag
2. You could clone the tag at the C terminus, just remove the stop codon and fuse the tag in frame
To select the tag you could read the articles below:
http://www.sciencedirect.com/science/article/pii/S1046592805000276
http://rd.springer.com/article/10.1007%2Fs00253-002-1158-6
In some cases the IP experiment could be replaced by a pull-down experiment with an appropriate resin
If you has further questions don't hesitate to contact me again
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