One protein has pI value is 6.34 and the other one has 6.41. I want to separate these protein,
As long as I tried, it seems to be impossible to separate those low pi value difference protein by Q sepharose.
How about to use size exclusion?
What are the sizes of the proteins? Are there any histidyl residues on their surface? What about their surface hydrophobicity?
The molecular weight of one protein is 58kDa (pI -6.34) and the other one is of 65.6kDa (pI 6.41) . No both proteins does not have histidine stretch on their surfaces. The protein of pI 6.34 has more hydrophobic patches than the other one.
You can try hydrophobic interaction chromatography to separate them.
You could try ion exchange "on steroids": chromatofocusing. This technique allows to separate proteins with minimal pI differences. http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-us/products/AlternativeProductStructure_17390/
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