I am working with a sweet-protein 'Thaumatin' (pKa ~ 11) which is secreted by genetically modified Pichia pastoris GS115. Since the protein-producing organism is grown in Basal salts medium, the sample retrieved from the bioprocess contains, the protein of interest, salts, cell metabolites, and other impurities. As a sample purification step before HPLC analysis, the proteins within the sample are precipitated using TCA and sodium deoxycholate.
Would you suggest a suitable buffer to resuspend the attained pellets before injecting the samples? The column used for the protein analysis is Zorbax 300SB and the mobile phase used are Acetonitrile and MilliQ water each containing 0.1%(v/v) TFA.
I have successfully resuspended TCA protein pellets using water, water/acetonitrile or 100 mM NaOH, prior to HPLC analysis. It really depends on the protein, or protein mixture.
Daniel Carneiro Moreira Thank you for the reply. I did some runs in the past after resuspending the pellets in MilliQ water. I was wondering whether changing it to water/acetonitrile or 100 mM NaOH improves the peak shape?
Hi Jewel,
It depends on proteins, but I usually prepare the protein sample in 20-30% acetonitrile/0.1% trifluoroacetic acid.
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