This is a peptide about 1KD in size. The commercially synthesized powder was reconstituted with a citrate buffer(pH 7) and the solution turns acidic, pH ~4. The assay requires a pH at 7 so some basic are added to make solution neutral, while the addition of extra basic interferes with the following assay?. So is there a way that I can dialyze this peptide? I know there are many commercial dialysis kit available, but just did find any that can work well for a 1KD peptide.
Thanks in advance for any suggestions.
Hi Wei,
We've stumbled across a similar problem while working with a tripeptide, and found that for our case ion exchange chromatography worked best (over failed attempts using size exclusion chromatography). If your peptide contains charged residues, anion or cation exchange chromatography may be suited for your needs. While the peptide can be eluted as pure (upon protonation/deprotonation which accompanies ion exchange), the final pH will depend on your specific peptide sequence and may still need to be titrated towards 7, if that pH value is far from the peptide's titration-equivalence point.
What type of assays are you planning to conduct after dissolution?
Depending on your test we may say added alkaline agents can affect the test or not.
That's for sure the molarity and type of the alkaline agent is also considerable but wondering that you say pH 7 is needed for further assays then why did you prefer citrate buffer at pH 7 instead of a stronger buffer system at higher pHs?
Physical desalting/ buffer exchange is hard to apply to these MW's. Interaction based exchanges using resins like WCX/WAX/RP etc. and solid-phase ext. cartridge formats may help.
Omar is right. For peptides, reverse phase HPLC is the go-to technique. If you don't have access to HPLC, you can use manual solid phase extraction (syringe and SPE cartridge) to achieve the same physicochemical effect.
İsmail Emir Akyildiz Thanks for your suggestion. The peptide will be use for ITC, for which any buffer mismatch is trying to be avoided. That is why I have to make the peptide in neutral without any extra addition of alkaline. We did use buffer like HEPES that is stronger at pH 7, but it did not help to solve the problem. We have limited access to the resins. Peptide purification with a solid cartridge will also introduce acid residual as TFA is used based on my experiences.
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