Phospho-Specific Antibody Production

The most common peptide modification used for polyclonal antibody production is the phosphorylation of amino acids; which is accomplished immunizing host animals with a short synthetic peptide containing the phosphoryl (PO(3)) group on the residue of interest. Phosphorylation events are integral to cellular regulation functions including:

• Activation of enzymes and receptors
• Cellular transcription
• Signal transduction
• Cell signaling networks

The Process:

Custom phospho-specific antibody production can ensure that the peptide mimics the natural conformation and activity found in the protein. Phosphorylation often results in functional changes in the activity of a protein, and having antibodies recognizing the phosphorylated state (as well as the corresponding non-phosphorylated state) can be integral for studying protein pathways.

There are three residues which can be phosphorylated:

• Serine (S)
• Threonine (T)
• Tyrosine (Y)

Custom phospho-spedific antibody production against a peptide containing a phospho-residue is a bit more complicated than traditional peptide antibody production. The animals can produce antibodies against additional epitopes in the peptide sequence, as well as produce antibodies that do not contain the modified phospho-residue. While a pool of phospho and non-phospho antibodies can be difficult to work with, if purified from the anti-serum the non-phospho antibody can be very helpful for studying protein pathways. This service included in our custom phospho-specific antibody production packages 3PN and 4PN.

Factors for Success:

• Length: use a peptide with a length of 12 residues or less. Length is important due to the number of epitopes available to the host animal’s immune system. The minimum length of an epitope is 6-8 amino acids; using a sequence with 12 or fewer residues ensures that the phosphorylated residue will be included in all antibodies produced.
• Position of Phosphorylated Residue: place the phosphorylated residue in the middle of the peptide sequence. This increases the probability that all antibodies produced against the sequence will have epitopes including the phosphorylated residue.
• Peptide Purity: use a peptide of >90% purity; all of our phospho packages include a phospho peptide of >90% purity to ensure that the antibodies are produced against the exact sequence of interest.
• Antibody Purification: even using a short, high purity peptide the polyclonal anti-serum will contain antibodies with and without the phosphorylated residue. Phospho-specific and non-phospho antibodies can be separated and isolated through a two-step purification process:
  • Two immuno-affinity columns are constructed, one using the phosphorylated peptide and one using the corresponding non-phospho peptide.
  • Anti-serum is passed over the non-phospho column to isolate antibodies that do not contain the phosphorylated residue.
  • The flow-through (which contains the antibodies with the phosphorylated residue) from the non-phospho column are then run over the phospho column.

  This process, which is included in our custom phospho-specific antibody production packages 3PN and 4PN, isolates both phospho-specific antibodies and the non-phospho antibodies for your protein. The corresponding antibodies can be used separately or in conjunction to study your protein’s regulatory pathways.

Additional Phospho-Specific Antibody Production Information:

Phospho-specific antibody production procedure: an overview of the process involved in custom phospho-specific antibody production.

ProSci offers four custom phospho-specific antibody production packages to assist in your research. PDF copies of the phospho-specific antibody production information sheet and phospho-specific antibody production order form are available.