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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