Long stretches of hydrophobic amino-acids, such as transmembrane domains of membrane proteins, are generally considered to be very bad antigens because of their very poor solubility in water and thus accessibility.
Nevertheless, there are some examples to the contrary, for instance antibodies against TM domains of GPCRs or of viral envelope proteins. I would appreciate any insights into the possibility to raise such anti-hydrophobic antibodies, and ways to do so.
you can always try the conventional way with mice immunization. you can also look in to phage display library of antibodies although not many successes with membrane proteins. in GPCRs i think they ued nanobodies raised from llama immunization
Dear Pierre
The peptide solubility problem can be resolved by coupling the hydrophobic peptide to a immunogenic carrier such as keyhole-limpet hemocyanin (KLH) which is a pretty good peptide antigen carrier. However, hydrophobic sequences are very poor antigens and tend to elicit non-specific antibodies. The average antibody binding pocket interacts with about 4-6 amino acid residues, if my memory serves me right. Specificity of antibodies in binding to a peptide antigen is highly dependent on hydrogen bonding and polar interactions. Many of the transmembrane domains have more polar regions at their terminal ends for interaction with the lipid head groups. So, if you hydrophobic peptide have a sequence of 4-6 amino acids with a few polar residues, you can maybe use a tandem repeat as you peptide antigen.
Hope it helps.
Regards
Marina
We ordinarily do not recommend developing antibodies to hydrophobic regions, unless it is an absolute requirement for very special purposes. It is so much easier to generate antibodies to hydrophilic regions that the effort to generate an antibody to a hydrophobic region must be worth the extra effort. We specialize in generating antibodies to 10-15aa peptides in goat. For hydrophobic peptides we would need to dissolve the peptide in the presence of DMSO in order to link it to its carrier protein.
Also linking the peptide to the affinity column will need some extra attention. Please contact me to further discuss.
What we did is that we linked the peptide to a spacer and attached that to a tripeptide and finally to a carrier protein. We generated antibodies in mice, rabbits and sheep.
Check out this paper
Universal antibodies and their applications to the quantitative determination of virtually all subtypes of the influenza A viral hemagglutinins
http://www.sciencedirect.com/science/article/pii/S0264410X08012322
Antigen preparation, prepare your peptide with some ligand then this introduced in to rabit to get good antibodies. Try several times
Thanks to all for sharing your ideas. Seems to be quite a trial and error process, with no magic trick, nor guaranty of success.
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