These are the results of an old experiment, which I still find hard to explain.
In cloned proteins with 6xHis, sometimes one can't detect 6xHis in the folded form, since it is buried in structure and might be unavailable for detection antibodies.
After denaturation we can usually detect protein through 6xHis with western blots.
In our lab a colleague of mine cloned a protein, and couldn't detect it with the same anti 6xHis antibody in western blot, although a band in the right size appeared strong in coomassie staining (checked vs. 0 time of expression in production process). After performing ELISA or Dot blot we could see good staining.
Protein was detected in the folded form, and not denatured, eventually it was also shown to be present in samples.
Can anyone offer an explanation?
You detect a denaturated protein in the first case (in the native form the His-tag is unavailable) and native in the second (via ELISA). Was this result replicable? Is it possible that the His-tag is cleaved in the second case? Are there any other possibilities to not detect the tag in the denaturated form (western)?
We had in another case cleaving of the tag, but then you can't detect with anti His antibodies in any of these methods and we had to obtain protein specific antibodies.
In this case the His Tag was detected in ELISA but not western, the opposite of what you would expect and this is what is puzzling.
Maybe most of the protein does not have the His tag, but a small population still does, so it is a mixed population of proteins. Since the Elisa is more sensitive, it is detecting the small population of proteins that still has the His tag.
Marcia, you broaght up a pretty simple and interesting idea which we'll need to check out.
Thanks
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