Antibodies that discriminate between the various states of methylation of an amino acid in a protein, is an increasingly asked for type of PTM specific antibody. Although the strategy for the production of methyl-specific antibodies in principle is the same as for making acetyl- or phospho-specific antibodies, it is no trivial task.
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 methylation 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 methylated peptide for immunisations is of course crucial, but the importance of the actual quality of the methylated peptide cannot be stressed enough. A common misconception is that peptide quality should be judged by HPLC purity alone, and that this purity will be the same in any analytical HPLC setup. Not true, and when it is not possible to separate the target from the impurity, a large percentage of the wrong peptide may inadvertently be used for immunisations. There is much to be said about producing high quality antibody generating peptides, but the simple matter of the fact is that this step is pivotal for the successful outcome of your project.
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 methylation state. These cross reacting pan-antibodies will recognise your protein regardless of whether it is methylated or not. These need to be removed from the sample, which is why a non-methyl peptide has to be made, with which pan antibodies are absorbed. 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 for example can differentiate between a methylated and a non-methylated protein, but not between the mono and di-methylated states. The potential risk for this is minimised by making the immunisation peptide with the best reagents possible.
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. In our custom methyl-specific antibody service, you will benefit from our commitment to providing excellent quality and customer service.
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