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Antibodies For All Your Research

Production and Services

PHOSPHO-SPECIFIC ANTIBODY PRODUCTION

Phosphorylation is the most common amino acid modification used for polyclonal antibody production. Phosphorylation events are integral to cellular regulation functions including:

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

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 main amino acids which can be phosphorylated:

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

Phospho-specific antibody production is a bit more complicated than traditional peptide antibody production. Phospho-specific antibodies are produced by immunizing the host animals with a short synthetic peptide containing the phosphorylated amino acid. The animals can produce antibodies that not only recognize the modified amino acid, but also antibodies that do not recognize the modification. In order to isolate phospho-specific antibodies a two-step purification process is needed. This service included in our custom phospho-specific antibody production packages 3PN and 4PN and is described further below.

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' 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 antiserum 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.
    • Antiserum is passed over the non-phospho peptide column to isolate antibodies that do not recognize the phosphorylated amino acid, leaving behind antibodies that recognize the phosphorylation.
    • The flow-through serum from the non-phospho peptide column is then run over the phospho peptide column, isolating antibodies that recognize the phosphorylation.
    • 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:

Download the Phospho-Specific Antibody Production Procedure (PDF) for an overview of the process.

ProSci offers four custom phospho-specific antibody production packages to assist in your research. To view our package offerings, download the Phospho-Specific Antibody Production Information Sheet (PDF).

Ready to order? Just fill out the following Phospho-Specific Antibody Production Order Form (PDF).

If you have any questions regarding our phospho-specific polyclonal antibody production packages or need peptide antigen design assistance please contact us at abservices@prosci-inc.com, or by calling toll-free 888-513-9525.