Hello, folks!
I have a patient's serum, and I have synthesized a few peptides that I know have a strong bond to my target antibodies. After getting the Ab-Peptide to form a complex in a microplate, I need to separate them and capture those antibodies for subsequent sequencing.
How should I proceed to separate them and capture those antibodies?
Well, you can pull down the Ab, if you first conjugate your peptides. You can conjugate them to a sticky protein, like serum albumin, and plate them as a capture antigen, like for ELISA. Or as Jiang suggests, conjugate them to paramagnetic microbeads. Either way, dilute the serum 1/10 in PBST, add it to the capture antigen, and give it 30-60 minutes to stick. Swirl it occasionally, if you're using beads. Then rinse several times with PBST to reduce non-specifically bound background.
You can then elute most of the Ab from the capture peptides by adding a low pH buffer. The most popular cocktail is 0.1M Gly-HCl pH 2.1. After a few minutes remove the liquid using a pipet, and neutralize the pH of the eluant by addition of 1M Tris-HCl, pH 8. Then you can dialyze or desalt the protein on a column. Note that you will elute many different Ab proteins, and this method generally will *not* elute the Ab with the tightest binding.
For volumes, 100 uL should be plenty for loading, washing, and eluting most microplate wells. Use 10x bead volume, if you're using beads. For neutralization, use 20 uL Tris per 100 uL Gly. Fortunately, room temperature is fine for these reactions.
Unfortunately, you're not going to like the results you see on sequencing the protein, for at least two reasons. First, as I mentioned above, your patient's serum will contain dozens or perhaps even hundreds of different Ab proteins that are pulled down by each peptide. On a protein sequencer this heterogenous input will generate a uselessly heterogenous output that looks like loud noise. Second, Ab are infamous for bearing blocked amino termini, so the Edman chemistry for protein sequencing won't work unless you first enzymatically deblock the chains, which will generate more noise.
I agree with John and Jiang that the best way to conjugate the synthesized peptides to a carrier protein then add antibodies and elute like an affinity chromatography procedure
Hello! Thanks for all the answers.
John Carter , about the issues you listed at the end of your answer, do you think I'd still face the first issue you mentioned if I had many copies of only one peptide (around 20 bases-long) that I know bonds to my target antibody?
The first issue at the end? Yes, your antiserum will contain polyclonal Ab. You will not observe a consensus protein sequence unless you have a monoclonal Ab.
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