Does anyone have an IHC protocol for detection of a his-tagged protein in mouse tumor tissue (cryo, not paraffin)?
I'm trying to stain for a protein that contains a 6His tag and accumulates in tumor tissue (human tumor xenograft grown in mice). I used the M.O.M. blocking kit from Vector Labs to block endogenous mouse IgG, then incubated with the mouse penta-his primary antibody. The secondary antibody used was the Vector Labs Impress HRP-anti-mouse antibody. The problem is I get really high background signal, even in the negative control (no primary antibody). It seems the only species of penta his antibody is mouse derived, so maybe someone knows a source of rabbit, goat, or donkey penta his antibody?
Thanks,
Jason
Hi, Jason.
The high background of IHC-Fro for tumor tissues may be caused by the antibody's species. So, you can try non-mouse derived antibody, such as sheep Anti-6X His tag® antibody (ab84162) for IHC-Fro staining. Or you can stain the tumor tissues by Dye-labeled anti-6Xhis antibody, and observe the results by Confocal or fluorescence microscope. The fluorescence method can avoid the effects of endogenous peroxidase which can also result in high background signal.
Another reason may due to the specificity of the anti-His tag antibody. Generally, the anti-6X His tag antibody not only react with 6X his tag, it may react with the proteins with the domain which consists of a high proportion of histidine. So, you can analyse the specificity of the antibody by running Western Blot for tumor cells or tumor tissues.
I hope this helps.
best
Kelong.
Hi Jason,
You can use a Rabbit anti HIS-tag Ab like:
http://www.abcam.com/6x-his-tag-antibody-chip-grade-ab9108.html
and combine it with other polymer secondary system like DAKO EnVision or Zytomed (we use Zytomed on mouse tissues regularly)
Epitopes rich in histidine are your trap anyway. Other strategy is to change the tag
I hope you find it helpful.
Regards,
Ivan
anti-Tag antibodis are always good for a surprise because the sequence context may influence the accessability of the tag. For some proteins a certain Tag antibody works perfectly and fails if you use another protein with the same tag.
However I would also go for the recommended rabbit antibodies and give them a try.
Best,
Henrik
Thanks very much to all of you for the advice. I did manage to find a goat polyclonal anti-6his antibody from Abcam that worked well so I will continue using this in the future. I couldn't get the M.O.M. kit to work from Vector labs.
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