We would like to create antibodies against a very hydrophobic region of a protein. The KLH linked peptide itself is absolutely insoluble and can only be solubilized by adding hydrophillic amino acids to the end or beginning. But still it needs to be pre dissolved in DMSO.
We immunized rabbits with these peptides and only got a weak but robust immunereaction verified by ELISA against the peptide. Now we have the problem that we don't see any specific signal in a Western-Blot with the full-length protein.
I quess the problem is that hydrophobic regions are not prone to stimulate an immune reaction but there is no possibility to use a different region of the protein.
Does anybody have experience with antibodies against hydrophobic peptides and has any good recommendations on how we could get a well working antibody?
Hydrophobic peptides are problematic.
1. Is the peptide a trans-membrane sequence? - these are very hydrophobic and highly conserved resulting in cross-reactivity with other proteins.
2. Size of peptide - if small little or no immune reaction. If too long may form secondary or tertiary folding making it difficult for antibodies to recognize native protein. Use peptide longer than 12 amino acids but smaller than 21 amino acids. Check that the sequence is unique using a program such as Expasy. Also check that your sequence is antigenic (programs available on the net). You can also send me the sequence of the peptide so that I can check antigenicity for you. Alternatively send me the complete protein sequence because then I will be able to select potentially good antigenic sequences using Bioinformatics.
3. I normally make monoclonals against hydrophobic peptides and then select antibodies with high affinity & specificity.
4, Remember to treat samples with mercapto-ethanol and boiling before gels to denature protein. When you do your Western blot incubations make sure you have detergent in the antibody buffers. A 0.05 % Triton-X100 works well.
Good luck
Hydrophobic peptides are problematic.
1. Is the peptide a trans-membrane sequence? - these are very hydrophobic and highly conserved resulting in cross-reactivity with other proteins.
2. Size of peptide - if small little or no immune reaction. If too long may form secondary or tertiary folding making it difficult for antibodies to recognize native protein. Use peptide longer than 12 amino acids but smaller than 21 amino acids. Check that the sequence is unique using a program such as Expasy. Also check that your sequence is antigenic (programs available on the net). You can also send me the sequence of the peptide so that I can check antigenicity for you. Alternatively send me the complete protein sequence because then I will be able to select potentially good antigenic sequences using Bioinformatics.
3. I normally make monoclonals against hydrophobic peptides and then select antibodies with high affinity & specificity.
4, Remember to treat samples with mercapto-ethanol and boiling before gels to denature protein. When you do your Western blot incubations make sure you have detergent in the antibody buffers. A 0.05 % Triton-X100 works well.
Good luck
A valid alternative could be to link your peptide to NH- or COOH-teminus of an hydrophylic protein. For example, linking hydrophobyc peptide to E2 to protein of PDH complex (you can read some articles of Piergiuseppe De Berardinis) or to another protein as GST (by using pGEX 4T-3 plasmid) could be a good idea. Then you could purify by HPLC chromatography or by GST pulldown.
Dear Mr. Pool thank you for your fast answer.
1) The peptide is no trans-membrane sequence but highly hydrophobic and unstructured.
2) Both Peptides we used were 17 AA long and the sequence is more or less unique. The sequence similarity to other proteins is less then 55%. The antigenicity indeed is a problem the predicted value was not that high but as mentioned it is not possible to use any other sequence of the protein as we would like to distinguish isoforms which only differ in this region.
Is there a any method to stimulate an immuneresponse against such a sequence?
3) Monoclonal antibodies would be a good idea due to the possible selection of highly affine antibodies but I quess it will largely exceed the planned costs. But I will definitely keep it in mind.
4) That is what I usually do.
Dear Mr. Schiavone,
thank you for your answer. The approach with E2 sounds interesting. I will have a closer look to the method.
In addition to Edmund's remarks, it should be appreciated that a successful antibody raised to a hydrophobic region will not work on the entire protein, unless this protein is forced to expose its relevant hydrophobic region for the antibody to bind to. This can be fixed with enough non-polar detergents around. But the real challenge is to get a high enough titre in the first place, very difficult with hydrophobic regions.We made use of multi-antennary peptide, rather than linking it to a carrier protein.
Good luck
We have some experience about. If you use urea (< 3 M) for denaturation of the hydrophobic peptide before immunization, the resuting antibodies will recognize your electroblotted protein or it immobilized on ELISA plates. Rabbits are not affected by immunization with urea < 3 M.
Thank you for those good ideas. As the rabbits are already immunized denaturation of the peptides with urea won't be possible but maybe it could be easily used for an boost.
I am not sure if I understood the approach with non-polar detergents correctly. Would you reccomend to add the detergent to the primary antibody solution? Do you have a protocol for this (e.g. detergent and range of usable concentrations)? This would be really interesting to test.
Thank you very much!
I do not know if re-immunization with the denatured peptide will works. The question is that a hydrophobic peptide takes a secondary structure in a physiologica buffer; therefore your antibodies were produced against the folded peptides and do not recognize lineal epitopes on the whole protein but against the peptide itself. Sorry, the only way is to start again. We had this problem time ago and it was the only solution.
We add 0.2 % Tween 20 in all the buffers used for ELISA or immunoblotting.
Thank you very much for the clarification. I didn't think about that problem. I will consider the denaturation for a possible new immunization.
Daniel,
Ana Maria is correct. Any hydrophobic region will assume a structure to minimize its hydrophobic residues in an aqueous environment. Then, using non-polar detergents combined with the antibody are used to probe the blot with the antigen / protein of interest. I think of Tween-20 up to 0.5%, 1%NP40, 1%TX100 or a combination of them.
Just for the information of others that may face the same problem:
The immunization worked out in the end. We were immunizing the rabbits for roughly 700 days in the end with a switch of the peptide every couple of immunizations. The peptide region that we wanted to generate an antibody against was always kept the same but we were using different linker regions to keep the hydrophobic peptide soluble. As we started to get antibodies against this soluble region we switched it. The affinity purification of the antibody was then performed against a peptide with another linker region.
The detection of the purified protein of interest in a WB works well with 0.5 % Tween 20 or 1 % TX100.
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