His-tagged antigens - Must the tag be removed?? (Feb/21/2006 )
I am currently having troubles cleaving the His-tag from a NiNTA purified protein as it precipitates when dialysed into the buffer required for cleavage.
To overcome this problem it has been suggested that I dont remove the his tag and use the tagged antigen for polyclonal antibody production anyway (in a precipitate for resuspended in PBS).
However I am concerned that the immune response will be to the tag, hence produce his tag specific antibodies.
I have conducted searches on the web and some companies say the his-tag does not matter (opposed to other tags such as gst) yet other companies say it must removed. (Very confusing!)
I am interested to know if anyone has succeeded (or not) in recovering specific antibodies to the antigen rather than the tag when injecting a tagged-antigen (his or other type). Any opinions on the matter are also more than welcome!
first my disclaimer, i havent ever made an antibody..
however, i would think that you should be okay leaving the tag on... if you want a polyclonal you could remove his-tag binding antibodies with a column before using it to probe your blot etc.
Although to be fair, i thought this was true with GST too.. in fact, I thought sometimes you added peptides to GST on purpose b/c the peptide isnt large enough itself to elicit an immune response...
Just my thought... HTH