A protein may come in a number of isoforms. An example of this is the PITX2 protein, where there are at least five isoforms. Obtaining en isoform specific antibody may be as easy as indentifying an exon that is unique for the isoform of interest. Conversely, when you require an antibody that targets all isoforms, the antibody should target an exon which all isoforms have in common. This is straight forward and is accomplished with a simple anti-peptide antibody. In many cases however, it is only possible to discriminate between two isoforms by looking at unique exon bridges.
In such cases, the strategy for successfully making an antibody that specifically targets a certain isoform, and not the others, is essentially the same as for making specific antibodies to fusion proteins, neo-epitopes, point mutations, or post translational modifications; i.e. immunise with a peptide that corresponds to the target sequence, and absorb cross reacting antibodies using peptides corresponding to the non-target proteins.
In contrast to other projects, designing peptides for antibodies for raising isoform specific antibodies, there is not an abundance of possibilities. The peptide has to span the exon bridge 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 join – 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.
Having a good immunogenic antibody generating peptide is essential, but it might not be sufficient. There is a possibility that the antibody will cross react with one or more of the other isoforms. This is why the resulting antibody should be tested against the corresponding sequences of similar exon bridges, if these exist. Removal of the cross reacting antibodies may then be attempted by absorbing them.
Designing a good peptide for immunisation 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 long and the short of it is that there is no room for corner cutting and that means that you need the better raw materials, you need the better synthesis strategy, and you need excellent quality control to match it.
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 scientific community research services, such as our custom isoform specific antibody service. Our commitment to providing excellent quality and customer service are things that you and your research project will benefit from as well.
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