I am performing experiments using synthetic cell-penetrating peptides in acute hippocampal slices. Our lab has verified the NLS-mediated nuclear delivery of a number of such peptides in the region of interest. We are interested in synthesizing a phosphomimetic peptide with aspartic acid (D) in the place of a phosphorylated serine residue – a widely used methodology. However, our inquiry is dependent on the use of a phospho-specific polyclonal antibody. That is, our antibody targets the phosphorylated serine residue in which we intend to mimic via D substitution.
Does anyone have experience using phospho-specific antibodies along with D-substituted exogenous peptides? Will the antibody bind to the peptide with aspartic acid substituted in the place of a phosphorylated serine residue?
Thanks!
Hi Spencer,
I don't think this is standardly performed and might vary from antibody-to-antibody. You'll likely need to test it. Presumably you can just preincubate some peptide with your antibody to see if has blocking ability for IHC or Western applications. Assuming there is a reason you can't use synthetic phosphorylated peptides. Probably most times the antibody will bind a phosphomimitic peptide...some even will bind a nonphosphorylated peptide although they are phosphospecific on the protein level.
There is another thread that talks about the utility of aspartic acid phosphomimetics and the last comment is someone asking why they see a huge signal with their phospho-antibody when they overexpress a S-to-D mutant:
https://www.researchgate.net/post/On_mimicking_Phosphoserine_does_it_really_work
Hi,
With regard to phosphospecific polyclonal antibody, I do not feel that it will be highly specific to or distinguish specific single amino acid substitution. There might be a small subset of antibodies in your stock that can be highly specific but they may not give you an observable distinction. In short, though we call them phosphospecific they are not completely that since polyclonal antibody can have antibodies of multiple specificities - to any part of the peptide used as antigen.
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