General guidelines for making Acetylated (Lysine) antibodies

Here is some general information on making anti-acetylated specific antibodies. As you know, it is not trivial to make acetylation specific antibodies. The choice of peptide is also limited since antibody needs to be generated to the acetylation specific-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 acetylation specific 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, acetylation specificantibodies. From our experience in making acetylation specific specific antibodies, we recommend the following:

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

2. Synthesize the non-acetylated peptide as control as well. We will test antibodies by ELISA against the non-acetylated and acetylated peptide to determine the extent of antibody cross reactivity. The non-acetylated peptide can also be used as an affinity support to remove the unwanted antibodies.
3. Use the purified acetylated peptides (>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-acetylated or acetylated peptide-Sepharose or both. This will become clear once we test antibodies by ELISA and your own testing.

General cost estimates

1. Std. peptide synthesis (up to 20-aa) and antibody (2 rabbit) package for up to 20 aa with affinity purification $2290.00 (cat # ABPEP-20UA).
Additional costs, as compared to non-acetylated antibody projects, are .
2. Acetylation and Purification of the peptide ($150 for per Lys-acetylation. Purification of acetylated peptide ($150 per peptide over the std. 70-85% pure peptides)
3. Synthesis of control non-acetylated peptide, ELISA and testing will add approx. $600 (15-20 mg, 70-85% pure, mass, hplc).
4. The affinity purification package is $695.00 (coupling of peptide to Sepharose, purification of antibodies (1 bleed), ELISA testing of antibodies, and supply of affinity support for later use). There will be two columns: control and acetylated peptide.
5. Additional animals. The cost of adding an extra animal (rabbit/chicken) is just $300 per animal when initiated along with the other animals. You can add as many animals as deemed necessary. We typically add 1-2 animals per project (total of 3-4 per project).
Total Cost for items #1-5 = $**** for 3 rabbits per project (add $$$$ per additional animal).
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