Antibodies that discriminate between the phosphorylated and the non-phosphorylated state of a protein, is one of the most frequently asked for types of PTM specific antibodies. These could be made as monoclonal antibodies but are more often produced as antigen specific affinity purified polyclonal antibodies. Regardless, and as with any anti-peptide antibody project, making phospho 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 phosphorylation 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 phospho 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 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.
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 phosphorylation state. These cross reacting pan-antibodies will recognise your protein whether it is phosphorylated or not. When developing hybridomas, this is taken care of in the screening process. For polyclonal antibodies, these need to be removed from the sample. A des-phospho peptide has to be made, with which pan antibodies can be removed. These pan-specific antibodies can be very useful and the extra collection, workup, testing and documentation, are well worht the effort.
There is a certain possibility that you end up with an antibody that more or less only requires the phosphorylated 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 phospho specific antibody should be tested against three peptides; the immunisation phosphorylated peptide, the non-phosphorylated absorbtion peptide, and an unrelated phosphorylated 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 phosphorylations. 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 phospho-specific antibody service include;
Baumann, Ursula, et al.
Disruption of the PRKCD-FBXO25-HAX-1 axis attenuates the apoptotic response and drives lymphomagenesis.
Nature medicine 20.12 (2014): 1401-1409.
T. Steinfeldt, S. Könen-Waisman, L. Tong, N. Pawlowski, T. Lamkemeyer, L. D. Sibley, J. P. Hunn & J. C. Howard. 2010.
Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii.
PLoS Biol 8(12): e1000576. doi:10.1371/journal.pbio.1000576
M.-J.Boucher, L. Selander, L. Carlsson & H. Edlund. 2006.
Phosphorylation Marks IPF1/PDX1 Protein for Degradation by Glycogen Synthase Kinase 3-dependent Mechanisms.
The Journal of Biological Chemistry, 2006:281, 6395-6403.
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