Hi Sonja,

I assume you would like to detect your protein of interest using antibodies against the His-tag. Since your tagged protein will be in its folded state do you know whether the His-tag is accessible to anti-His-antibodies?

From my experience, I would recommend a capture ELISA but that requires that you need two different specific antibodies. Unfortunately, I do not know about a commercial kit.

Best regards,

Christian

Have you googled it? 

I've found three companies comercializing either the antibody or the ELISA-friendly kit...

http://www.genscript.com/kit/L00436-His_Tag_ELISA_Detection_Kit.html

http://www.abcam.com/6x-his-tag-antibody-hrp-ab1187.html

http://www.cellbiolabs.com/his-tag-protein-elisa

If you were looking for some personal reviews on the quality of the kits themselves, from personal experience, then I can't help... Maybe it's a naive question or not-suitable for your lab, but why don't you consider column cromatography? - wouldnt that be the standard procedure?

Good luck!

Yep, I googled it but I was indeed hoping for some personal experience. :)

We want to quantify not just detect and use it to verify a allegedly protein specific ELISA (so far there are no antibodies that successfully specifically recognize our protein; plenty of them available but they only work reliably on transfected cells but not on fresh cells). Thanks for your replies so far!

I would recommend testing your serum by a simple dot blot technique just to make sure your anti-His is recognizing your expressed protein tag, as mentioned above you need to make sure it is exposed and not in the center of the protein. Once you know it is recognized, i think you could make a standard curve with your purified recombinant and measure in your sera. If the His-tag is not on the outside, maybe you could try some denaturing buffer (using 2-mercapto, DTT, Urea, Guanidine) to disolve your sample and then measure by ELISA.

Maybe you could take samples after your immunization protocol and check if your organism produced antibodies against your rAg and then produce your own specific antibodies, i did that experiment on my master, immunizing mice with pADN and weeks after collected blood and centrifuge serum, Abs worked fine.

Best regards, 

Cristhian Sández.

Hi Cristhian,

thank you for your reply! The problem is not in generating antibody, there are in fact a couple that do recognize the protein on transfected cells very nicely. Unfortunately none of them works to stain cells that are rT-PCR positive  in cryosections, paraffin embedded antigen-retrieval treated section or by flow cytometry  (even though one might be working on cells that have been extensively stimulated in presence of the antibody). So, there might be an issue with masking the epitope by its binding to a receptor or being mainly shed instead of membrane bound.

We will probably resort to western blot (working antibody available) to verify whether the protein we express in the mice is reliably recognized by the new protein specific ELISA we want to test. Initially we thought detecting his-tag by ELISA might be a good idea to also compare sensitivity of the assays but apparently it is not well established and I think it might not be worth the effort trying to set it up in our lab.

Best regards,

Sonja

Hi Sonja,

Just wondering if you were able to quantify your His-tagged proteins in serum using commercial ELISA kits?  I am in a similar situation here and looking for real world experience.

Regards, Anne

Hi Sonja and Anne,

May I know if you eventually decided on using any of the commercially available ELISA kit? Did you find your target of interest? Thank you.

Best,

ChiaLing