Could protein modification block an antibody epitope? - (Sep/12/2007 )

Hey everybody. I was wondering whether or not a protein modification such as Ubiquitination could block an antibody's epitope? What's happening is that whenever I do a western with a whole cell lysate, I see multiple bands (that are comparable in size to mono-ubiquitination) that do not seem to be non-specific, because the bands change in intensity according to treatment (in many different ways). But when I do an IP, I only get the one band I am supposed to get with the antibody. My reasoning was that, my protein was denatured during the SDS-PAGE so the antibody is able to recognize the epitope on the western, while the IP is done with a non-denatured sample so the antibody doesn't recognize the modified proteins' epitope, only the unmodified.

I am currently doing an IP with a denatured sample but don't have the results yet.


DOES THIS MAKE ANY SENSE TO ANYBODY ELSE??

Thanks for looking.

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I think that u can come to some conclusions after looking at the blot (IP with a denatured sample)

All the best

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is your antibody polyclonal or monoclonal?

was it made against native or denatured protein?

only one of the polyclonals target epitopes may be accessible on the native protein.

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It is a polyclonal antibody. Commercially available from Abcam. I have no idea if it was made against native or denatured protein.

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On the site Abcamin column Info you can open full product datasheet, where you will find information about nature of immunogen

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