When a point mutation within the protein coding region of a gene occurs, the resulting protein may have a single amino acid substitution. It is possible to generate an antibody that specifically targets the mutated sequence.
When wanting to obtain an antibody that differentiates between two very similar sequences, such as when a certain site is in its phosphorylated state and when it is not, it is obvious how very small differences in the sequence have a great impact on the resulting antibody. For these types of projects anti-peptide antibodies is the tool of choice. Peptide design, which is the basis for all anti-peptide antibody projects, will have a direct impact on the success of the project.
Of course, when designing peptides for antibodies that target epitopes spanning a point mutation site, we don’t have much liberty when it comes to choosing location within the protein. Nevertheless, even small changes in the sequence can make a significant difference. The idea is to to reduce the number of trivial epitopes – i.e. epitopes that do not contain the mutation – while having sufficient length to the peptide so that the yield of specific antibody is likely to be good. Furthermore, the peptide should be soluble, and we want a unique functional group that facilitates the conjugation to a carrier molecule. Sometimes the design is straight forward, sometimes this requires some careful tweaking in order to work.
Designing a good peptide for immunisations is of course crucial, but the actual quality of the peptide is also important. We'd like to think that we know something about producing high quality peptides for antibody generation - and we're happy to talk about it at great length - but here it suffices to say that these types of projects do require the better raw materials, the better synthesis strategy, and excellent quality control to match it. Just think of it: we want to target a point mutation. How then can we accept the use of a peptide that contains a percentage of deletion sequences or amino acid adducts?
A high quality peptide antigen is crucial, but the resulting polyclonal antibody is still likely to contain species that do not depend on the mutation site. These cross reacting pan-antibodies will recognise your protein whether it is mutated or not. If this is the case, then these cross reacting antibodies can be removed from the sample, either by absorption or blocking with the corresponding peptide of the non-mutated protein. These pan-specific antibodies can be very useful and although they require collection, workup, testing and documentation, they are an inexpensive by-product when using absorption rather than blocking as a strategy for obtaining the point mutation specific antibody.
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 that specifically recognise point mutations or post translational modification sites. Our commitment to providing excellent quality and customer service are things that you and your research project will benefit from as well.
Examples of references of customers using our custom point mutation specific antibody service include;
C. Spanaki, I. Zaganas,K.A. Kleopa & A. Plaitakis. 2010.
Human GLUD2 Glutamate Dehydrogenase Is Expressed in Neural and Testicular Supporting Cells.
The Journal of Biological Chemistry, 2010:285, 16748-16756.
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