My HA tag did not work, and its not because of the antibody!?

Question

I cloned an HA tag on the N-terminus of a vector and I sequence verified and concluded that the tag was there. And I also verified everything like the promoter was correctly in the vector and further more there is no frameshift. I transfected cells and I used an HA antibody to see my protein but the blot was blank!!! Just to make sure I used the original antibody of the actuall protein to locate it and it was clearly there! So what is going wrong. My protein is 55kda. Can an HA tag be cloned in an N-terminus? Can an HA tag form a secondary structure inhibiting antibody connection. Does cloning an HA tag make the protein insoluable with certain lysis buffers?

Answer 1

I haven't used HA tag, but here is a thought.
Could it be that your N-terminus is processed and the tag cleaved? Check the literature if there is some processing at the N-terminus of you protein. Have a look at commercial vectors to see if there is something different in the linkage between the tag and protein (maybe you are introducing a protease site)
If you are using bacteria you should remove the ATG from your gene and keep only one, in front of the tag, to avoid different initiation of translation.

A more simple thought is that the anti-HA has gone bad (or is not good to start with), or you are not using the proper secondary antibody for it.

If you were changing stability/solubity of your protein you wouldn't be seeing it with the other antibody. You are either losing the epitops by proteolysis or shifted translation or there is something going on at the antibody-epitope or primary antibody-secondary antibody interaction level.

Also for blots, proteins are denaturated so you shouldn't be worried about the sructure of the protein. Make sure the anti-HA you bought is suitable for blotting (denatured proteins) and not for the native state.

Personally I am not using tags. A friend of mine tried most of them and if I recall correctly he was rather disatisfied with HA and used flag and triple flag, though that was for in vitro transcription-translation experiments.