I could not find a solution/explanation for my problem, and really need some advice or suggestion. I have a cell line that does not express protein A of interest. I then transfect this cell line with untagged protein A, protein A tagged with HA, and protein A tagged with Flag separately (tag at C terminus). I ran western blot, denaturing condition with SDS (samples have SDS and are boiled for 10mins). I could detect the proteins by anti-Flag and anti-HA beautifully, at the right molecular weight. But when I use antibody against protein A itself, I can't detect any band for the tagged proteins, but I can detect it in cells that have untagged version. The antibody is monoclonal that has worked robustly. It does recognize the sequence at the C terminus where I added the tag. But I added 2 Gly between the terminus and the tag, under denaturing condition, I do not expect the tag to mask the epitope.
I can't tag the N terminus because it affects localization of the protein. Most of antibodies (and all good ones) in the market for this protein targets the C terminus. I am pretty confident that the protein is expressed, but I don't know how to make it show up using the antibody. Any help is greatly appreciated!
Hi
Maybe the active site of protein for ab is covered by tag or may be the natural conformation of your protein is chaned due to tag.
A possible reason for this phenomenon may be that the mAb epitope includes the carboxylate group of the C-terminal residue of the untagged protein, which is not present in the tagged protein.
It is unfortunate that the tag interferes with detection of protein A by the anti-protein A mAB, but it does not seem to be a serious problem, since you can still use the anti-tag antibody for this purpose.
Yes, I agree with the others. You answered your own question. Your results clearly say that the epitope is being masked by the tags. Science sometimes requires you to discard your own pre-conceived story in favor of the story told by your results. Better get used to it :)
The obvious way to fix this would be to use a polyclonal for detection. It should bind all over your protein, and if one side becomes inaccessible, plenty of others will still work.
Thank you all for your great advice. @Adam: your answer is the first to give me an explanation why an epitope can be masked under denaturing condition. I've done a lot of searching but could not find a mechanism for why this could happen. Thanks a lot!
I tried to search for an antibody recognizing other part of the protein, and there is only one in the market, but it reacts with human protein and has not been tested for mouse (i forget to say i work with mouse cells). All other antibodies recognize the C terminus, I guess because this epitope at C terminus has worked so well. I will try the human antibody and hopefully I will be able to detect it.
You already had several good answers. The only thing I would add, in response to your second message, is that it is often a mistake just to rely on what is 'available'. There is a built-in inertia about the alternative approach of either raising your own antibody or, if funds allow, getting one tailor-made commercially. The fact that it may take 3 months and you want your answers today or tomorrow tends to make people reject or postpone the decision to get their own antibody. ......Three months later they wish they'd raised the antibody! Even if you don't have pure protein, a synthetic peptide offers a good alternative and allows you to choose the region of the protein to be recognised.
Paul is correct: making your own antibodies is very rewarding, and every cost effective, although my experience using synthetic peptides is not so good!
But can you not simply cleanly eliminate the tag from the aminoterminal end of the protein? But it does seem like you would need to make sure that the protein's own initiation codon and down stream AAs are intact. Easy enough to fix by PCR it its not the case.
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