Hi researchers,
As the title shows, recently I made a construct with a 8xHis tag at N ternimal of HBx (154 aa). The voctor is pCDNA3.1+ and the sequencing result showed everything was correct.
I use both anti-His (sc-803) and anti-HBx antibody to detect the protein after transient transfect. For anti-HBx, I can detect a very clean band which means the protein is translated but for anti-His, I can not detect my 8xHis tag.
HBx is a viral protein and cannot be secreted so I don't think post translationnal modification cut this tag off. Is it possible if the antibody is called a 6xHis one, it cannot detect 8x or 10xHis tag?
Now I have the Ni-NTA column and am going to pull down 8xHis taged HBx and detect with HBx antibody, even in western blot I didn't see the His band. Am I correct that the longer the his tag, the higher affinity it has to bind Ni-NTA? 10xHis is better than 8xHis and 8xHis is better than 6xHis?
What is the principal of adding his tag? I see commercial one is only 6x or 10x, why don't have other numbers?
Thanks！
Do you have the a construct with 6xHis tag? Were you able to detect a protein with 6XHis tag with anti-6xHis antibody? Sometimes, it may be impossible to detect His-tagged proteins using anti-His tag antibody. Generally, it depends on the folding of your proteins. The His-tag might be buried deep inside upon protein folding. In this case, it will be difficult to detect the protein using anti-His tag antibody. It will be also difficult to purify the protein by IMAC.
I think the effect of the overall conformational structure of your protein will be more significant than the effect due to change in the number of His in the sequence of His-tag.
If your anti-His6 antibody can detect the protein with the His6 tag, it should also be able to detect the protein with the longer His tag.
Longer His tags should bind more tightly than shorter tags to Ni-NTA columns, allowing for a higher imidazole concentration when washing the column, hopefully reducing the amount of binding of untagged His-rich proteins in the extract.
Hi Alemu,
I only made 8His tagged protein. Yes, I'm afraid that the tag is buried inside so before pull down, I did the western blot. However, the protein is linearized before loading, it doesn't matter the natural protein is inside or not right?
Hi,
can you estimate from your anti-HBx-western blot how much protein you actually have in your cells? Note that not all antibodies have the same sensitivity, so your Hbx-antibody could just be better in detecting tiny amounts of your protein while the His-tag antibody needs more protein.
Do you have a positive control for the His-tag? Just to be sure that there is no problem with the antibody.
Another problem is that if you have very low expression, you might not have enough protein in your culture to get reasonable amounts purified using Ni-NTA, because you end up with a high amount of unspecifically bound proteins (also those directly or indirectly bound to nucleic acids, DNA binds quite well to Ni-resins).
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