Inja

The epitope is indeed discontinuous, I read the paper you mentioned, I was just concerned as to being told to detect a discontinuous epitope using western blot following denaturing electrophoresis. Yes I am working on the assumption that although continuous epitopes are easily recognised, discontinuous ones are not. Thanks, I have my answer Perhaps I will post on other threads, or become a contributing member, but that depends on how much time I have as I expect to be quite busy in the lab for the next few months. ciao

Well of course the individual amino acids don't break apart, but I guess what your saying is that the protein only partially unfolds, leaving many discontinous structures intact. I had always pictured SDS-based denaturation as more of a chain reaction in which the penetration of one SDS molecule into the protiens hydrophobic core opened up gaps that allowed for further molecules to associate with the polypeptide and so forth until the protein was completely unwound. This was of concern to me since I have been instructed to detect a certain protein molecule by western blot, using a monoclonal antibody which I know recognises a discontinuous epitope... Thanks for your help.

Is this not the purpose of the SDS-PAGE run before? To denture the protein and cover it in a uniform negative charge so that it migrates according to its mass, and then is not the protein used in the western blot subtracted directly from this same denatured sample? The epitopes would only not denature if they were continuous epitopes... Correct?

Something I can't quite grasp, How is it one can detect proteins in Western blot using antibodies, if the epitopes recognised by these antibodies have been denatured in the SDS-PAGE run beforehand? Are you just hoping that the epitopes are continuous or that they partially reform upon transfer to the membrane? Thanks