What is the goal of this step? What happens if we omit this step and load solubilized inclusion bodies on C4-HPLC column directly?
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It might precipitate on the column if not refolded. But if the c4 is run in acid, and it is still denatured, it might be ok. What is the reason for the DEAE step anyways?
Because just solubilized inclusion bodies contain many impurities and other bacterial proteins. If you will load this mixture directly onto C4 column without additional purification, I am afraid the column will die.
That makes sense. If the inclusion bodies aren't that pure, then it is a better to use the DEAE column first because it is cheap to replace. Just for my benefit, why do you need the c4 HPLC column? Are there very similar protein impurities that you are eliminating with this. In the paat, I used a huge HPLC column because the proteins I needed to purify we're so similar in nature.
Dear Marcia, thank you for your comment. We also use C4 column with big volume, although the peaks corresponding to our target proteins (slurp, lynx or snake neurotoxins, you can learn our other papers) always stand separately from other peaks. So we initially do crude purification and then use C4 column. The combination of these approaches always give us the protein samples with purity more than 95%. Moreover C4 column allows us to separate refolded and denatured proteins.
Thank you for the explanation. I did not know it can separate denatured from refolded proteins. Is this true for many proteins, that a C4 column can do this?
Dear Marcia, I think it does not matter whether your HPLC column is C4, or C18 one (it depends on the protein you purify), anyway you are able to distinguish correctly folded and misfolded species of the same protein. This is true for proteins containing numerous disulphide bonds... Dear colleagues, thanks a lot for sharing your expertise on this question!
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