Hello everyone,
I would like to use a biotinylated 5AA peptide to perform a Pull-down/MS experiment, to screening peptide-binding receptor proteins. But I'm not sure if this experiment will work, because 5AA peptide is hard to form secondary structures so their binding to the receptor may be instantaneous and difficult to capture.
Does anyone can give me some suggestions.
Wait for your warm-hearted reply.
Thanks!
You're probably correct in guessing that the receptor requires contact with more than 5 AA to achieve binding strong enough to pull down a receptor protein. My experience with monoclonal antibodies suggests about 1 in 8 of them binds to a short continuous peptide. Most require two or more (discontinuous) strands. Maybe you will be lucky.
Still, to make this work you will need to include a spacer between the biotin and the peptide. Common spacers include one or two epsilon-aminocaproic acid molecules, although I prefer to simply include the tetrapeptide SGSG. You can also buy biotin conjugation reagents that include a spacer.
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