I want to study truncated versions of a protein to analyse its domain specific function. So, for detection I need to put some tag at the C terminal as I have N terminal deletions. I have used HA and His tag. HA has worked fine but His hasn't. Is there a problem if I use 2 tags together ike Flag and HA or should I use individual tags?
When you say His-tag hasn't worked for you it was being used to purify the protein or as the target epitope in Westerns?
If it was for purification then make sure you have no (or Low) EDTA and reducing agents in your lysis buffer as these could affect the Ni-column/beads, also add a few 'flexible' amino acids (GGS, GSS, AS...) as linkers between the end of your protein and the his-tag to improve tag accesibility. If it was for Westerns unfortunately there are lots of useless anti-his antibodies out there so it could just be your detection method.
I don't see why you cannot use any combination of FLAG, HA, His, myc, StrepII etc. tags but it really depends on your use-purification or detection or both?
Dear Tias,
As far as i know, you can use both the tags together. i did use both flag and HA together for same protein in two different plasmids.. both of them expressed well and was able to be detect them by western blotting.
Hi Tias,
If the addition of the tags does not appear to cause any problems to your protein then having 2 tags at least keeps your options open should you have problems with a particular antibody (as happened here with your anti-his Ab). If at some point in the future you want to try to co-precipitate or purify your protein the presence of 2 tags may be very useful. I would keep them if they do not interfere with each other or your experiments. Obviously you only need to detect one at a time.
We use anti-penta his monoclonal from Qiagen that works well for us (with the right blocking solutions!).
Dear Tias,
i agree with nick, they do come handy in co-immunoprecipitation. infact you can check the formation of protein dimers..
Dear Tias,
the tag is from reverse primer..
the model system is yeast and its a haploid strain.. that's why to maintain two copies of the protein i used two different plasmids having two different selection markers so that cells can retain both the genes of same protein.
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