Technical Data
CS010-002
Custom Polyclonal Antibodies, Phospho-Specific
Program 1
Program 2
Program 3
Custom Services Storage: Shipping:
Custom Phospho-Specific Polyclonal 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 (PO3) 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:
The generation of phospho-specific antibodies 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)

The process of producing antibodies 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.

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 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.

Projects with Phosphorylated Residues:
One of the most common peptide-modifications used for antibody production is the phosphorylation of residues; this can ensure that the peptide mimics the natural conformation of the protein. Phosphorylation often results in functional changes in the protein, and having antibodies recognizing the phosphorylated state can be very important for studying protein pathways. However, the process of producing antibodies against a phospho-residue peptide a bit more complicated than traditional antibody production.

There are three residues which can be phosphorylated:
• Serine (S)
• Threonine (T)
• Tyrosine (Y)

Step 1: Synthesize two peptides
• Peptide #1 (non-phospho peptide): the peptide sequence of interest without the added phosphorylation on the residue of interest; require a minimal amount – 5mg should be sufficient
• Peptide #2 (phospho peptide): the peptide sequence of interest with the phosphorylation; requires at least 10mg of peptide

Step 2: Conjugate Peptide
• Conjugate the phospho-peptide to carrier protein, such as KLH

Step 3: Immunization protocol
• Immunize phospho-peptide conjugate into animal(s)
• Remove serum at scheduled interval

Step 4: Evaluate serum
• Serum contains a mixture of antibodies; some of the antibodies will have the phosphorylated residue, while some antibodies do not have the modification

Step 5: Purify Serum
• Separating the phosphorylated antibodies from the non-phosphorylated antibodies is performed by immuno-affinity purification, using two columns:
o Non-phospho peptide column: bind the non-phospho peptide to gel matrix for purification
o Phospho column: bind the phospho peptide to gel matrix for column
Serum containing a mixture of phospho and non-phospho antibodies serum
Affinity purified non-phospho antibody
Non-phospho column
Flow-through (contains phospho antibody)

• Run the serum through the non-phospho column. The eluted affinity purified antibody will contain antibodies without the phosphorylated residue. The flowthrough will contain a mixture of phospho antibodies as well as other antibodies present in the serum
• Run the flow-through through the phospho column. The eluted affinity purified antibody will contain the specific, phosphorylated antibody
• Use the phospho peptide to run an ELISA on the serum, phospho affinity purified antibody, and the flow-through resulting from the phospho column
Flow-through (contains phospho antibody)
Phospho column
Affinity purified PHOSPHO antibody
Program 1:
Anti-Sera Schedule:
* 8 week schedule
* 2 rabbits
* 2 pre-bleeds (5ml per animal)
* 8 immunizations (4 per animal)
* 6 production bleeds (3 per animal)

Phospho Peptide Synthesis:
* Up to 15 residues for phospho peptide
* HPLC purity of >90% on phospho peptide
* Includes 1 phospho residue (pS, pT, or pY)
* Allow 2 weeks

Conjugation:
* Conjugation of phospho peptide to KLH
* Allow 1 week

ELISA Report:
* On pkg. peptide(s) at 1st production bleed

Program 2:
Anti-Sera Schedule:
* 8 week schedule
* 2 rabbits
* 2 pre-bleeds (5ml per animal)
* 8 immunizations (4 per animal)
* 6 production bleeds (3 per animal)

Phospho Peptide Synthesis:
* Up to 15 residues for phospho peptide
* HPLC purity of >90% on phospho peptide
* Includes 1 phospho residue (pS, pT, or pY)
* Allow 2 weeks

Non-Phospho Peptide Synthesis:
* Up to 15 residues for non-phos peptide
* HPLC purity of >70% on non-phos peptide
* For non-phos ELISA and purifications

Conjugation:
* Conjugation of phospho peptide to KLH
* Allow 1 week

ELISA Report:
* On pkg. peptide(s) at 1st production bleed

Program 3:
Anti-Sera Schedule:
* 8 week schedule
* 2 rabbits
* 2 pre-bleeds (5ml per animal)
* 8 immunizations (4 per animal)
* 6 production bleeds (3 per animal)

Phospho Peptide Synthesis:
* Up to 15 residues for phospho peptide
* HPLC purity of >90% on phospho peptide
* Includes 1 phospho residue (pS, pT, or pY)
* Allow 2 weeks

Non-Phospho Peptide Synthesis:
* Up to 15 residues for non-phos peptide
* HPLC purity of >70% on non-phos peptide
* For non-phos ELISA and purifications

Conjugation:
* Conjugation of phospho peptide to KLH
* Allow 1 week

ELISA Report:
* On pkg. peptide(s) at 1st production bleed

Affinity Purification:
* 25ml serum purified over each column
* Column construction for pkg. peptides
* Each animal’s serum purified separately
* Includes re-usable columns

Example of a 2 rabbit custom antibody schedule:
Week 0 (Pre-bleed) Pre-immune serum collection ~ 5ml serum/rabbit
Week 0 (Immunize) 200ug/rabbit in Complete Freund's Adjuvant (CFA)
Week 2 (Immunize) 100ug/rabbit in Incomplete Freund's Adjuvant (IFA)
Week 4 (Immunize) 100ug/rabbit in Incomplete Freund's Adjuvant (IFA)
Week 5 (Bleed) 1st production bleed (available to ship at day 36) 20-25ml serum/rabbit
Week 6 (Immunize) 100ug/rabbit in Incomplete Freund's Adjuvant (IFA)
Week 7 (Bleed) 2nd production bleed 20-25ml serum/rabbit
Week 8 (Final Bleed) 3rd production bleed 20-25ml serum/rabbit
Intended for research use only. Not for use in human, therapeutic, or diagnostic applications.