Does denaturing proteins lead to loss of epitopes?

Question

I am doing an experiment where I have to do both Immunohistochemistry and SDS-PAGE. I am assuming that the native conformation of the protein is maintained in the IHC. But during the blot, we heat the protein (at least in our lab) at 95^oC in mercaptoethanol to denature the protein by breaking the disulphide bonds thereby destroying the tertiary structure.

My question is when we are using the same antibody against the protein in the tissue (intact for IHC) and isolated (denatured for SDS-PAGE) protein, how can it bind both? So, the antibodies bind to the epitope based on the AA sequence or the 3D conformation? Wouldn't there be a lot if cross reactivity if it binds just based on sequence (since you could have the same sequence in many proteins, having the same conformation is much less likely)?

Answer 1

Short Answer - If the same antibody works both for IHC and SDS-PAGE it either means it is monoclonal against a surface linear epitope or a polyclonal antibody.

Long Answer -

Starting off, there are broadly two kinds of antibodies based on the type of the epitopes. The epitope can either be linear or conformational. Here's a couple of pictures that should clarify.

Now coming to the techniques, in IHC although there is little denaturation, one can expect the conformation to be intact. On the other hand, SDS-PAGE requires the protein to be drawn into linear structures so that they can pass through the gel.

When the manufacturer says that the antibody is monoclonal and can be used both for IHC and the blot (manufacturers do mention the compatibility of the antibody with different techniques and species), it is most probably derived against a linear peptide on the surface of the protein. This means that it would work for IHC (since it needs a surface epitope) or SDS-PAGE (which needs a linear one). May you keep in mind that, a surface epitope can either be conformational or linear. Monoclonal Abs are produced by the hybridoma technique.

On the other hand, if the antibody is polyclonal, it is much more likely that this set contains some IgGs that bind linear epitopes and some that bind surface epitopes. Thus a polyclonal antibody is much more likely to work on both SDS PAGE and IHC. It is also somewhat easier to produce, since it is essentially only purified antisera from an animal that was immunized with the antigen of interest.

Answer 2

Many but not all B cell epitopes will be destroyed by denaturing. Those that are conformational will be destroyed, but there are also many B cell epitopes that are linear, based on amino acid sequence only without strong conformation dependence.

Also, depending on your IHC protocol, there's often a fixation step that involves a certain amount of denaturation, though rarely as drastic as in immunoblotting. Conversely, some proteins are capable of at least some restoration of native conformation after they bind to your blotting substrate.

Bottom line, it's unpredictable, but it works often enough to make it worth trying.