I am trying to set up indirect ELISA using recombinant protein and antibody.
The problem is the epitope of my antibody is "N-terminal".
The most of recombinant protein I found is "tagged with GST or 6-His in their N-terminal".
In this case, could my antibody bind to this recombinant protein?
Should I have the GST or His removal process or change my antibody which has different epitope?
Hi there,
You can't anticipate from the start because you don't know the effect of the GST on the accessibility of the epitope (you have no structural clue about how both will arrange in the fusion protein).
Dear Liger, thank you for answer. I am based on mechanical engineering, so it's not familiar to me. In my opinion, the tagged state of N-terminal means there is no available area to bind other molecule (which means my antibody). In this case, could my antibody (which has epitope of N-terminal) bind to protein if the N-terminal of protein is exposed to antibody?
Dear Schmitt,
Is it meaning the His is no problem to use but the GST is not guaranteed?
Dear Alsaedi , thank you for your kindness.
I found the availability of FLAG or His tagged protein to ELISA.
Maybe this is meaning availability on GST tag.
Thank you for your help.
The meaning of my first answer is that you don't know whether the GST will actually hide the epitope recognized by the antibody or not. Antibodies are not specifically raised against N-ter or C-ter epitopes, they may react on sequence lying right in the middle of the protein primary sequence (it means the sequence is sufficiently exposed and acessible to react with specific antibody).
To Liger, thank you for your explanation. I misunderstood about epitope. I think it kinds of long train with linker, and there is no available area or linker to antibody if there already tagged.
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