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Poor reproducibility during peptide synthesis

Poor reproducibility during peptide synthesis - Peptide Forum

Poor reproducibility during peptide synthesis - Peptide Forum. Ask and discuss questions on peptide protocols, custom peptide synthesis, peptide identification and peptide sequencing.


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Old 04-18-2008, 10:30 AM
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Question Poor reproducibility during peptide synthesis



I am now synthesizing a cyclic peptide (32 amino acide residues) contain a disulfide bond formed by SH of two Cys. My workmates have synthesized the peptide before. The problem they came across was that each batch they did showed poor reproducibility. RP-HPLC analysis of the linear precursor showed a small peak ahead or behind the main peak. Since I have synthesized many peptides, almost RP-HPLC of every peptide I synthesized showed this similar phenomenon. This great affected the yield of the product.
Does the phenomenon I described above normal or not? What can i do or what protocol can i take to avoid the formation of the small peak ahead or behind the main peak?
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Old 04-18-2008, 12:05 PM
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Default Re: Poor reproducibility during peptide synthesis

The peak before can be the cyclic and the one after can be dimer. Try to use slightly acidic conditions when dissolving the peptide (0.1% TFA) and dont let it stand in solution for long time before purification and after.

Last edited by adrianandreica; 04-18-2008 at 12:08 PM.
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Old 04-18-2008, 06:28 PM
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Default Re: Poor reproducibility during peptide synthesis

Doing the cyclization reaction at low peptide concentrations may help too. I use 0.1 mg/ml peptide in 0.1 M sodium acetate pH 8.5 and it worked fine for almost all of the peptides I've synhtesized.
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Old 05-05-2008, 11:46 PM
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Default Re: Poor reproducibility during peptide synthesis

Dear yuanhenglidlut,

I assume you are talking about the purification of your linear peptide. You should simply collect the side-products and analyze them. It really could be cyclic and dimeric peptide as adrianandreica mentioned. It even might be something different, e.g. isopeptides. Without analysis of the side-products one can not really help you.

We often see small peaks before and after the product - usually I am happy if they stay small. How can small peaks influence the overall yield?

Good luck,

AplaGen
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