I have purified my recombinant protein of 55 KDa using Ni-NTA affinity purification method. As I got two other non-specific bands (one in higher molecular weight and another in lower molecular weight than my desired protein), I subjected them for another purification step through sephadex G-75 column. After gel filtration, the non-specific higher molecular weight protein was removed. But the non-specific lower molecular weight protein is still eluting along with my desired protein. Can any one suggest me some tips to get rid of the non-specific one?
Try to use hydroxyapatite resins for both intermediate purification and/or polishing steps. Multi-mode interactions will catch your impurity with at least one provided interaction (hydrogen bonding, CEX, ion exclusion or calcium affinity, etc..) You may try hydrophobic and IEX coupled resins also for purification.
It is also possible to increase the SEC resolution by decreasing the injection volume, by varying flow rates and column temperature but in the case of very identical MWs you should go through for the molecular interaction.
Emir...
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