HPLC Iso-Eluotropic strengtht for IPA [August 25, 2004]
Posted: Sun Aug 29, 2004 8:44 pm
By Greg Flugum on Wednesday, August 25, 2004 - 09:02 am:
I work in a university proteomics core lab. I had a suggestion for cleaning hplc columns (C4,8,18) of proteins and peptides to use 50-75% IPA/Water as opposed to 100% ACN. Does any one know the iso-elutrophic strength of IPA vs. ACN or MeOH? I can not think of any drawback to using 100%IPA except that the solvent strength might be too high to solublize peptides & proteins.
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By HW Mueller on Wednesday, August 25, 2004 - 11:29 pm:
There have been extensive discussions on this, in short: Most proteins are precipitated by ACN, MeOH, IPA.... Proteins differ immensely from ech other, some are dissolved by ACN/H2O or Alc/H2O, others need chaotropic substances (urea, etc.), still others detergents (including polyethers like PEG); pH is very important. In extreme cases you may have to reduce S-S linkages, thus, a mixture of dithiothreitol and Li-dodecylsulfate has been used as a last resort in this lab. Concentration plays an important role also, for instance, the wrong amount of detergent will give you a neat precipitation. A book on protein handling/purification is a good companion in this.
I work in a university proteomics core lab. I had a suggestion for cleaning hplc columns (C4,8,18) of proteins and peptides to use 50-75% IPA/Water as opposed to 100% ACN. Does any one know the iso-elutrophic strength of IPA vs. ACN or MeOH? I can not think of any drawback to using 100%IPA except that the solvent strength might be too high to solublize peptides & proteins.
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By HW Mueller on Wednesday, August 25, 2004 - 11:29 pm:
There have been extensive discussions on this, in short: Most proteins are precipitated by ACN, MeOH, IPA.... Proteins differ immensely from ech other, some are dissolved by ACN/H2O or Alc/H2O, others need chaotropic substances (urea, etc.), still others detergents (including polyethers like PEG); pH is very important. In extreme cases you may have to reduce S-S linkages, thus, a mixture of dithiothreitol and Li-dodecylsulfate has been used as a last resort in this lab. Concentration plays an important role also, for instance, the wrong amount of detergent will give you a neat precipitation. A book on protein handling/purification is a good companion in this.