Does anyone have any experience with this? I have a mixture of five proteins, the one I want purified out is ~10 kDa with a pI ~4.5. I have a strong anion exchanger: 150mm x 4.6mm, 4um, 80A pore size. I don't think it is designed for small molecules but it has similar specifications to other columns that are marketed as peptide or small protein SAX columns.
My question is what type of buffer can I run if I do not want my protein to denature (although the structure indicates it is very stable, I don't want to find out down the road that I was wrong)? Does anyone have a protocol that they've used for a similar separation, successfully?
Thanks in advance.
Most proteins and peptides 'prefer' a buffer like PBS (phosphate buffered saline).
you most use buffer with pH higher than 4.5.I think 0.1 M phosphate buffered pH 7 - 8 will be good for your separation .
Phosphate will compete for binding with your protein, especially at 100 mM conc. Moreover pbs uses a high concentration of NaCl that depending on the ligand density could also affect binding.
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