General guidelines for making phosphopeptide antibodies


Here is some general information on making anti-phosphopeptide specific antibodies. As you know, it is not trivial to make phospho-specific antibodies. The choice of peptide is also limited since antibody needs to be generated to the phospho-peptide at a certain location and surrounding amino acids (10-15 aa). This region may or may not be the best from the antigenic point of view. However, most phosphorylated peptides are likely to be hydrophilic and exposed at the surface of the protein (good for making antibody). ADI can also assist you in advising the selection of peptide and check peptide specificity by doing a BLAST search. Please send us the name(s) of the target proteins and chances of its similarity with other related members if any. This info will assist us finding the most specific peptides.
One can take certain measures to maximize the chances of producing useful, phospho-peptide specific antibodies. From our experience in making numerous phosphopeptide specific antibodies, we recommend the following:

1. Peptide length should be between <15 AA (. It doesn’t matter where the Phospho-amino acid is but you may want to avoid phospho-peptide at the very N or C-terminus. Longer peptides are likely to produce antibodies to the non-phospho amino acids as well.

2. Synthesize the non-phospho peptide as control as well. We will test antibodies by ELISA against the non-phospho and phosphopeptide to determine the extent of antibody crossreactivity. The non-phospho peptide can also be used as an affinity support to remove the unwanted antibodies.

3. Use the purified phosphopeptide (>90-96% purity as opposed to std 70-85% for regular peptides) for antibody production. High purity assures the removal of most impurities or truncated peptides that may affect antibody production.

4. We have seen a great difference in animal response. It is not unusual to find that a given animal has better specificity than the rest. In general, animals also differ in their response to a given antigen even if all procedures are done at the same time and inoculated with the same antigen. Therefore, we suggest additional animals (use at least 3 animals instead of std 2) per project to select the animal with the most specific antibody response.

5. In general It will be necessary to affinity purify the antibodies over the non-phosphopeptide or phosphopeptide-Sepharose or both. This will become clear once we test antibodies by ELISA and your own testing.
General cost estimates for anti-phosphopeptide Antibody Projects:
 

 

 

 

Services 

 

 

1

 Peptide Synthesis

    

2

Peptide Synthesis & ELISA

  

 

 3

 Synthesis of control non-phospho peptide (15-20 mg, 70-85% pure, ELISA and testing of all animals

 $***

 

 4

 Addition of more animals (2 are included in the std package) @$300 per animal

 $***

 

 5

 Affinity purification package (coupling of peptide to Sepharose, purification of antibodies, ELISA) using the control peptide

 $***

$***

 

Total for  Items #1-5

 

 $***

 ** If peptide contains more than 1 phospho-amino acid then the cost will be $* per residue. Typically, 2 phosphoresidues per peptide can be accommodated. It is not advisable to have more 2 phosphoresidues per peptide.

Total Cost for items #1-5 = $*** for 3 rabbits per project (add $300.00 per additional animal). Using these proven strategies, we have successfully produced phosphopeptide antibodies.

Std. peptide synthesis (up to 20-aa) and antibody (2 rabbit) package with peptide upgrade and affinity purification (cat # ABPEP-20UA).

Additional Services (optional);  $***   2   Phosphorylation and Purification of the peptide ($*** for per Tyr, Ser, Thr phosphorylation)   $150 each  

Upgraded purification (>95% pure) of the phosphopeptide   $.