Intact epitopes in Western Blot

[Inja]

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

[ecoli]

why are you assuming the epitopes will be denatured?

[Inja]

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?

[ecoli]

Well they don't really denature, in the sense that the residues of the protein break apart. SDS surround the proteins with negative ions, so that the proteins lose their unique tertiary structures.

 

This could cause them to lose their functional activity, but they aren't degraded to a point where the target antibody can't bind to them.

 

Note though, that even if a protein partially degrades, an antibody can still (potentially) bind to the protein, though it does depend on the antibody/protein. Also, the antibodies are probably less specific in their binding than you think. If you don't blot your membrane (and even if you don't) you'll get background 'noise' and possibly cross-reactive bands.

[Inja]

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.

[CharonY]

Actually SDS treatment will have effects on the efficiency of subsequent antibody assays. In case of linear epitopes the binding efficiency might be enhanced. Discontinuous epitopes, however are a tricky matter. In most cases denaturing electrophoresis is not recommended in such cases. Sometimes it does work, possibly due to renaturation. I also believe that strong disulfide bonds can stabilize the epitopes, depending how close the involved aas are within the protein.

So in short, Western blots are not really suited for most discontinuous epitopes.

I actually recall there being a paper a year or so back in which surprisingly discontinuous epitopes were detected in Westerns. Maybe I can dig it up.

 

Just to clarify a point though, the epitope to be detected is clearly a discontinuous one? Or has the AB just been initially raised against one?

[ecoli]

CharonY - it sounded like the OP was asking if SDS caused continuous epitopes to become discontinuous. I don't think this happens, but maybe that's not what the OPer was asking.

[CharonY]

Actually to me this part here:

Are you just hoping that the epitopes are continuous

implied that the it was assumed that continuous epitopes are detectable but the question was what happens if it's not.

[Inja]

Actually SDS treatment will have effects on the efficiency of subsequent antibody assays. In case of linear epitopes the binding efficiency might be enhanced. Discontinuous epitopes, however are a tricky matter. In most cases denaturing electrophoresis is not recommended in such cases. Sometimes it does work, possibly due to renaturation. I also believe that strong disulfide bonds can stabilize the epitopes, depending how close the involved aas are within the protein.

So in short, Western blots are not really suited for most discontinuous epitopes.

I actually recall there being a paper a year or so back in which surprisingly discontinuous epitopes were detected in Westerns. Maybe I can dig it up.

 

Just to clarify a point though, the epitope to be detected is clearly a discontinuous one? Or has the AB just been initially raised against one?

 

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

[r_bo_99]

I'm very weak in immunology, but when an antigen is encountered in vivo it is handled by the immune system in one of two different pathways: cross-linking or presentation by an APC (Antigen Presenting Cell).

 

If your detecting antibody was derived from the latter pathway, then the protein had already been denatured by the APC when the antibodies were developed, therefore denaturing with SDS should be the same as denaturing by the APC.

 

Bottom line: if your protein of interest is viral or self-protein, and the antibody was developed in a different species, then it should be recognized by the antibody, whether it is contiguous or not. Doesn't that seem correct?

 

PS I have zero experience with Westerns, this is a conjecture on my part. Rock on, Randy

[CharonY]

Actually you are right to consider how the antibodies were produced in the first place. It is e.g. a difference if the protein or only the relevant peptides were presented (or if the design was first done in silico). I assume that in this case the antibodies in question have never before been used in a Western? Of course one can try doing a non-denaturing gel, though the resolution would be rather bad.

 

Anyway, APCs do not denature proteins (as antigens) in a similar way as SDS does, but they degrade it. So in the end they would present peptides. A discontinuous epitope of that antigen has to remain, per definitionem, intact. Otherwise it would not be an epitope, nor would it be considered discontinuous.