I’m trying to purify a fusion protein by two step purification STREP-6HIS. In my construct I would also like to introduce a HA taq because there are not good antibodies in my protein. I wonder if it possible to introduce an HA taq not at the terminal ends (NH or COOH), but consecutive to the HIS.
Es: HIS-HA-Protein-Strep
I would like to visualize the HA taq protein by Immunocytochemistry.
Dear Elisabetta,
the HA-tag can also be placed in between other tags. We constructed a double (-protein-HA-Strep) and a triple tag (protein-HA-Strep-Protein A) and in both cases the corresponding fusion proteins were detectable in immunoblot analysis.
Hi, Elizabetta, People have put GFP tag between two domains of a protein to avoid perturbation of a protein's function associated with N- or C terminal tagging. See Christina Lehner's work on Drosophila CID protein. Elaine Dunleavy et al. (2012, PLoS Biol) used the same construct to study CID in Drosophila animals, and only this internal GFP tagged CID protein can functionally replace the wild type protein, whereas the N- or C- terminally tagged CID protein can't rescue the lethality/sterility phenotypes of CID mutant. By the same principle, it is possible to insert an HA tag internally to a protein of interest, but it always has to be tested case by case.
Yes I agree with the above. That is the principle of TAP tag purification (tandem affinity purification) that is very useful, among other applications, for protein complex purification and identification by mass spectrometry.
I agree with Brian, strep should be good enought.
My two cents: in my experience, HA is quite a tricky tag, I would go for myc rather than HA.
The only anti-HA antibody I found to recognize the HA epitope within a protein (not on the N- or C-term is HA.11 from Covance. The HA epitope in one of my constructs is placed in an unstructured loop (position 235 of a 432aa protein) and I tried many antibodies to detect it by immunofluorescence. Only the HA.11 clone 16B12 works. I had some noise when using the antibody at 1:100 however it is very clean if diluted to 1:1000.
I tried to find a hybridoma, because I use the antibody for many applications, however it is not easily available.
I've used the anti-HA from Sigma, and although it works great for WB, signal for immunofluorescence was neliglible, and it was N-terminal alone.... Different people on different labs and reagents also reported the same problem. So maybe you find that it works when you check by WB, but it fails in your assay.
Rosario, I had the same experience with the Sigma antibody, where it works for western blotting and does not work for immunofluorescence. The same is true for the very popular Roche HA antibody mentioned by Shujuan, where it only works for WB when the HA does not have a free N- or C-termini. Again only the HA.11 has worked for me. If anyone knows of another that works I would certainly like to try it.
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