I have several his-tagged recombinant, purified proteins that are being detected in varying degrees by differentially his-tagged antibodies. I understand that sometimes part of the tag can be hidden, so that a 6xHis-tag antibody may not be able to pick up a tag where only 4His residues are available. What I am unclear on is why (this happened yesterday) a 6xHis-tag antibody would pick up a 6xHis tag well, but a penta-His tag antibody or tetra-His tag antibody used to detect the same protein would give no signal?? All experiments are done under non-denaturing conditions so proteins are in their native state. Thanks!
Hi Becky, in my opinion, it should be conformation and low affinity issues. Have you tried purifying under denaturing conditions?
Good luck.
Thank you for your replies. I have tried some Abs from different manufacturers, but with the same results. I have not done any denaturing conditions, because the assay that I am working on requires the protein to be in its native state. I just think its crazy that if I can get a signal from a 6xHis Ab (which should mean 6 His residues are available/not hidden) the 5xHis Ab would not be able to detect it. It's not even like I got less signal than the 6xHis Ab, I got NO signal. Do you think this could still be an affinity issue? Perhaps I need to look deeper into the nature of anitbody-epitope interactions. There is also a paper where they had similar problems with Epo. They claim to also have these issues under denaturing conditions but I don't believe it because its possible the proteins are still partially folded and they needed to add Gu-HCl or urea to their gel conditions.
I agree that His epitopes are often hidden especially under native conditions. Though it might be that at least one of the commercially available anti-His antibodies bind to epitopes containing the surrounding amino acids (RGS) not to polyHis. Are your antibodies from Qiagen?
my penta His antibody is from Qiagen, and the 6xHis antibody is from Thermo Fisher. Somewhere in the literature for Qiagen it mentions that this antibody is great because it doesn't require additional amino acids next to the tag. Do you think the Thermo Fisher 6xAb could be raised against HHHHHHRGS or something, and that's why it is not being picked up? For example, if my protein only has a His tag but not those extra residues?
Have you noticed a difference between Qiagen and others?
Yes, that's what i was thinking. But I m pretty sure that one of the Qiagen antibodies has the RGS (GS=BamH1 site) epitope but they label it as RGS. i havent used the Roche but it would be worth checking exactly what that antibody binds to.
Update: I wasn't able to track down the epitope that these antibodies are raised against. It may be proprietary information for the companies. I am now trying to understand how the tag can be partially "hidden". Is this due to some ionic interaction between the tag residues and protein residues? It seems to me that the tag itself should not really have tertiary structure, and should be exposed to the solvent for the most part. When looking at protein structure in pdb or pymol, I wonder if I should be looking at the water accessible regions for portions where the tag may not be water accessible, or some other category like a space filling model. Can anyone comment?
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