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I would like to purify my peptide having cysteine residue by using HPLC. It is known that thiol containing peptides undergo oxidation and form disulfide linkages. In order to prevent it I used proper cocktail solution during cleavage from the resin. However, I am still worried what if my peptide undergo oxidation during HPLC purification? How can I prevent it?

I searched in research gate, but I couldn't find suitable answers.

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  • $\begingroup$ One way is to add reducing agent to the running buffer. I don't know of another way. $\endgroup$ – Buck Thorn May 20 '19 at 9:05
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    $\begingroup$ What reducing agent I can use? Any experience? $\endgroup$ – Science123 May 20 '19 at 9:26
  • $\begingroup$ This might be useful as a comparison: agscientific.com/blog/2019/04/… Also, I recommend searching a little more, it's not an uncommon problem. $\endgroup$ – Buck Thorn May 20 '19 at 9:33
  • $\begingroup$ Most HPLCs have degassers, so oxygen tension in the apparatus should be low and oxidation should be minimal. You can inject from a TCEP (or DTT or BME)-containing sample, as recommended by @CuriousTree, and add these same agents to your fraction collector's tubes ahead of time, but you shouldn't need to add them to you chromatography buffers. $\endgroup$ – Curt F. Sep 3 '20 at 22:14
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I dont know about HPLC column tolerance to various reducing agents, this would need to be checked. However, in biochemistry we routinely add reducing agents to avoid disulphide-bridge formation in the proteins.

Overall there are three main reducing agents used, to avoid this problem. The choice of which one to use depends on your needs. They include:

  • TCEP: Considered the golden standard, it is relatively odourless compared to the other reducing agents. Once you have dissolved it, it should also remain very stable over time. Typically used at a 0.4 mM concentration.

  • DTT: Cheaper than TCEP, although I have heard that it is not as stable over time

  • 2-Mercaptoethanol: The absolute cheapest, stinks like crazy. Typically to a final of 5 mM.

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Rather than worry about reducing agents in the HPLC column, why not oxidize your cysteines with Ellman's Reagent and reduce them after purification?

As long as you use an excess of Ellman's Reagent, it should completely oxidize the thiols and prevent disulfide bond formation.

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