As regard western blot, which of the two types of antibodies will detect the total amount of protein, either phosphorylated or not?
It is possible to make a polyclonal or monoclonal that will detect the protein independent of phosphorylation. "Monoclonal" just means that every antibody in the vial is identical. Therefore a monoclonal recognizes one very specific epitope. If that epitope happens to contain a phosphorylation site, then maybe antibody binding will be affected. Similar for other post-translational modifications. If you make a polyclonal antibody by immunizing a rabbit with full-length protein, there will likely be antibodies that bind different parts of the protein. So if one epitope is subject to post-translational modificiation blocking antibody binding, other antibodies will still bind their epitopes. You can also make polyclonal antibodies against small peptides. These may behave more like monoclonal antibodies in the sense that they all recognize the same 10-12 amino acids that may or may not have a phosphorylation site in the middle.
The best is probably to have 2 different antibodies (monoclonal or polyclonal) that detect different epitopes. Often you can ask the company producing the antibody for the amino acid sequence of the immunizing peptide and check to see if there are post-translationally modified amino acids present.
It is possible to make a polyclonal or monoclonal that will detect the protein independent of phosphorylation. "Monoclonal" just means that every antibody in the vial is identical. Therefore a monoclonal recognizes one very specific epitope. If that epitope happens to contain a phosphorylation site, then maybe antibody binding will be affected. Similar for other post-translational modifications. If you make a polyclonal antibody by immunizing a rabbit with full-length protein, there will likely be antibodies that bind different parts of the protein. So if one epitope is subject to post-translational modificiation blocking antibody binding, other antibodies will still bind their epitopes. You can also make polyclonal antibodies against small peptides. These may behave more like monoclonal antibodies in the sense that they all recognize the same 10-12 amino acids that may or may not have a phosphorylation site in the middle.
The best is probably to have 2 different antibodies (monoclonal or polyclonal) that detect different epitopes. Often you can ask the company producing the antibody for the amino acid sequence of the immunizing peptide and check to see if there are post-translationally modified amino acids present.
Hi Mark
I have another question, please
if the immunizing epitope is 20 amino acid length, does this 20 a.a are sufficient to escape the post transitional phosphorylation?
And if the immunizing epitope Is 130 a.a would it better?
If the 20 amino acids contain a known phosphorylation site (see phosphosite.org) then you might be concerned. In general most people don't think about this, however. Immunizing with full length protein or a large fragment of the protein might help you have many epitopes recognized by the polyclonal antibody mixture. I have seen one case where even this approach resulted in a non-phospho-specific antibody.
The 20 a.a sequence contains a known phosphorylation sites, therefore, I think using the polyclonal ab against 130 a.a would be appropriate. Am I right?
There's no way to know until you try. Finding an antibody that recognizes the protein by western blot that it is supposed to is the first challenge.
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