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Phospho Antibody Production

The process of producing antibodies against a phospho-residue is more complicated than traditional antibody production using peptide immunogens. Phospho-specific antibodies are generated using peptides containing one or more phosphorylated amino acids. There are three residues which can be phosphorylated, Serine (S), Threonine (T) and Tyrosine (Y).

For phospho polyclonal antibody production:

To produce antiserum against Phospho-Peptides includes synthesis of phosphopeptides, conjugation and immunization of rabbits. In many cases, one would need to do affinity purification with a phospho-peptide column. Sometimes, one would also need to cross absorb the antibody with a non-phosphopeptide column in case there are some anti-non-phospho protein antibodies in the antiserum. In this case, synthesis of matching non-phospho peptides is required to make the negative-selection columns. Affinity purified, cross-absorbed polyclonal antibodies that are specific for the phosphor-peptides are usually required for downstream assays.

For phospho monoclonal antibody production:
In comparison with polyclonal antibody production, monoclonal antibody production against Phospho-Peptides is more straightforward; we just use the Phospho-Peptides to immunize the mice, and use Phospho-Peptides to screen for positive hybridoma clones. After that we use non-phospho-peptides to do negative selection. This negative selection is required [although widely forgotten] since peptide phosphorylation [or protein phosphorylation] is never 100% complete.

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