Antibodies that discriminate between the acetylated and the non-acetylated state of a Lysine residue in a protein, is an increasingly asked for type of PTM specific antibody. As with any anti-peptide antibody project, making acetyl specific antibodies requires that one thinks quality in every step of the production.
In contrast to other projects, designing peptides for antibodies that target a post translational modification we don’t have much liberty when it comes to choosing location within the protein. The peptide has to span the acetylation site of course, but even small shifts in sequence can make a significant difference. We want to reduce the number of trivial epitopes – i.e. epitopes that do not contain the PTM – while having sufficient length to the peptide for the antibody production. The peptide needs to be soluble, and we want a single facility for the conjugation to a carrier molecule. If possible, we want to avoid including potential PTM sites other than our target.
Designing a good Lys-acetyl peptide for immunisations is of course crucial, but the actual quality of the peptide is also important. There is much to be said about producing high quality antibody generating peptides, but the simple matter of the fact is that in order to obtain a high quality peptide you need the better raw materials, you need the better synthesis strategy, and you need excellent quality control to match it.
Having a good immunogenic antibody generating peptide is essential, but the resulting material is still likely to contain antibodies that do not depend on the acetylation state. These cross reacting pan-antibodies will recognise your protein whether it is acetylated or not. These need to be removed from the sample, which is why a non-acetyl peptide has to be made, with which pan antibodies are adsorbed. These pan-specific antibodies can be very useful and although they require collection, workup, testing and documentation, they are an inexpensive by-product.
There is a certain possibility that you end up with an antibody that more or less only requires the Lys-acetyl amino acid, pretty much regardless of what amino acids surround it. It is a situation that is not easily remedied – if possible at all – but at the very least you should know about the problem, if it exists. This is why any acetyl-specific antibody should be tested against three peptides; the immunisation acetylated peptide, the non-acetylated adsorption peptide, and an unrelated Lys-acetyl peptide.
Innovagen was founded in 1992, at a time when anti-peptide antibodies were not as common as they are today. Since then we have provided the research community with numerous antibodies targeting epitopes spanning post translation modifications such as acetylation of Lysine. In our custom acetyl-specific antibody service, you will benefit from our commitment to providing excellent quality and customer service.
Examples of references of customers using our custom acetyl-specific antibody service include;
S. Schlottmann, H. V. Erkizan, J. S. Barber-Rotenberg, C. Knights, A. Cheema, A. Üren, M. L. Avantaggiati, J. A. Toretsky
Acetylation Increases EWS-FLI1 DNA Binding and Transcriptional Activity
Front Oncol. 2012; 2: 107.
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