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?
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