I am working on expressing a protein that has a C-terminal His tag. I want to find out whether anti 6xHis antibody and anti-6x C-terminal His antibodies will give me different specificities.
Robert: Both antibodies were raised against 6-His peptide and doesn't care N- or C-terminus. But different Abs from different vendors do react differently in terms of sensitivity. So, check out yourself.
I conjugated anti-His antibodies with HRP to improve their specificity and sensitivity. For details, check out PMID: 25900303 or download from my RG.
Typically you need different epitopes to get different specificity, cMyc, HA or Ctag are good smallish epitopes with better antibodies than most His tags. In my hands the HIS.H8 clone is one of the best anti His antibodies.
6X His tag is nothing but His-His-His-His-His-His polypeptide that can be fused to any protein either at N-terminal or C-terminal. The terminals will be different in the N- and C-terminal tags. However, the antibodies agains 6X- His tag should recognize both N- and C- terminal tags provided they are exposed in your target protein.
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