I am facing with a perplexing situation in which there are two proteins both identifying correct protein sizes in immunoblot by polyclonal antibody (monoclonal doesn't exist in market) but it is probable that they both reacting with sth specifically in IHC and IF that we dont know its identity but it is very important for us to know potential cross reacting protein because of nature of experiment . I tried to get cross reacting protein x with IP with known antibody but no success.
Any creative method you know that be of help here?
Thanks
Hi Sepideh
I did not catch if your proteins have same MW or cross-react with antibody since having different MW?
If it is not a problem with unspecific epitope recognition by the antibody :
In the first case it could be isoforms of your protein of interest
In the second case it could be the same or a degradation/precursor maturation product of your protein.
@patrickrouimi Thanks for your reply. actually my 1st concern is the same as you mentioned.unspecific epitope recognition by the antibody if that is the case how I can investigate this? the interesting thing is that if the unspecific epitope be super similar to original protein I think that needs investigation(maybe unknown isoform?). How one approaches this as a matter of fact I'm not yet clear
Hi Sepideh
The first thing you may investigate is if the two proteins have same origin (one is the maturation product of the other by clivage or other). You should extract them from protein bands obtained by gel chromatography and analyze them by mass spectrometry proteomics (sequencing). Then you will know if they just belong to a proteins familly and then are different proteins or if they are isoforms of the same gene. I just remind you that an epitope (especially with polyclonal antibodies) is often related to secondary structure instead of simply to primary structure of the protein.
good luck
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