His tagged antigen - Does the tag need to be removed? (Feb/22/2006 )
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 form 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!
up to my knowledge and experience histidine tag does not serves as a strong antigen to raise antibodies. unfortunatley we cant exclude the possibility of getting anitbibodies against histidine tag.
(try to use your polyclonal antibody to detect other protein whihc is of not your interest but with histidine tag, then u will come to know what extent your PAb is specific to histag chain)
in fact the ratio between antibodies against his tag and your protein of interest will be very high (1:100 this ratio is not calculated but assumed) so you donot have worry too much about histag chain.
i used to produce monoclonal antibodies by using histage protein. only once i got 2-3 poor titre clones which produced histag Ab out of a hundred clones.
i hope u understood the experiment what i suggested
u r welcome for comments
We have used his-tag purified antigens to raise polyclonal rabbit antisera many times without issue.
Thankyou both for your comments... you have reassured me to go ahead with the process.