I am fairly new to ion exchange chromatography, and I am trying to optimize conditions to purify a protein having a pI value 8. I am using an anion exchanger (Q) in pH 6.0. I am aiming to separate my target by having majority of the impurities bound to the resin and my target flowed through during loading (unbound part). I succeeded in having most of the impurities bound, but what I am confused of is that I still find considerable amounts of my target to be bound to the column when it was not supposed to bind.
My POI has a pI of 8, thus it will be having a positive charge at pH 6 buffer and will not bind to anion exchange resins (in my understanding), but it turns out to still bind to it.
Can anyone please correct me if I misunderstand this principle? Your ideas will be much appreciated. Thanks^^
You're right, with a pI of 8 one would not expect retention at a pH of 6. But proteins are complicated molecules - there could be an attraction between the resin and unpaired electrons on some of the amino acid residues, and/or a certain amount of non-ionic attraction. Also, are you using a strong or weak base anion exchanger? If the former, that may at least partially explain what you're observing. What are the impurities you are trying to remove? Salts? Other proteins? I'm wondering why you chose to capture the impurities and not the protein of interest.
Alan Gabelman, Thanks! Yes I am using a strong base anion exchanger. I'm trying to remove other proteins from the sample. I have tried an anion binding condition for the POI previously using gradient salt elution, but based on the result, the impurities separated in this condition is more than with the POI bound. Just the problem is that some amounts of the POI is still bound even at lower pH. Also, the next step that I will do will use a cation exchanger so it is ideal that I do not have any salt in my sample.
I am still doing tests and comparing results though, so I will still probably try the anion binding using a different condition ie. pH elution probably. I just got curious as to how this binding is possible.
Richelle,
I am assuming that your impurities bind more strongly to AEX than POI, but the POI still binds somewhat- is that correct?
You may want to try the weak anion exchanger. Strong exchanger is still nearly completely ionized at pH 6, hence it doesn't loose any binding capacity. It is your POI that is affected by the lower pH much more. By switching to weak AEX you can affect the resin (ligand) as well.
If impurities bind strongly to aex you should be able to explore 1- elution of POI with low salt while the impurities are retained or 2- spiking load and having column at that low salt EQ to allow POI flow through.
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