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What epitope is my antibody recognizing? - (Jun/15/2006 )

I have an antibody (Ab3) that recognizes a protein X on cell surface. Pull-down experimets with an antibody recognizing a known epitope (Ab-cyto) on the same protein is not recognized by the Ab3 on an SDS-PAGE gel. On the other hand pull-down with Ab3 is recognized on SDS-PAGE with Ab-cyto i.e. the Ab3 works fine as a pull-down antibody, but it dosn't bind to a denatured epitope. Now to my question - from this set up I can not be sure that the epitope is a single-protein target. The Ab3 might bind in a groove produced when protein X interacts with an unknown protein on the cell surface... Is someone still following my thoughts? How should I do to be sure that it is a single protein target? Any suggestions?


It should be indicated on the datasheet of your Ab whether it is suitable for WB or not. It looks like it recognizes only the native form of your protein. If the other one can recognize the native form as well as the denatured, you could try and do a native gel (i.e. neither SDS in the gel or in the buffer) and see if they recognize the same band.


The antibody is produced in house using a pull-down with Ab-cyto as the epitope i.e. there is no datasheet. Yes, the Ab-cyto recognizes both native and denatured epitope and I tried the approach to run native gel. However, I have never run native gels before and it wasn't a success... See my question posted just a few minutes ago. I will try with different pH in sample and loading buffer, but feel insecure about the technich. Is there some other way to do this?



your Ab recognizes a conformational epitope.
It means that your antibody will only recognize the native protein.
the aminoacids that are recognized are localized on separate regions of the protein that are brought closer while the protein is refolded, and are separated when you denature the protein.
I don't think that the binding of your Ab will depend on an unknown protein interacting with.
Yes you might need to do some native gels. (or ELISA, I don't know what's the purpose of your experiments)