HomeFAQsI want to rebind MBP to the amylose column, but the maltose must be removed. Can this be done by dialysis?
FAQ: I want to rebind MBP to the amylose column, but the maltose must be removed. Can this be done by dialysis?
Dialysis does not work very well to remove maltose from maltose-binding protein. This is a general phenomenon of binding protein/ligand interactions; after the free ligand is gone, ligand that is released from the binding site usually finds another binding site before it encounters the dialysis membrane. We have determined empirically that binding the fusion to a chromatography resin and then washing away the maltose is much more effective. Standard chromatography (e.g., DEAE) is a preferred separation step, since it can separate the TEV Protease and MBP from the protein of interest if immobilized metal affinity chromatography is not an option. In case MBP co-elutes with the protein of interest, we include a large volume washing step to remove the maltose before starting the salt gradient. This way, the mixture can be run over an amylose column afterward if necessary. Alternatively, we recommend utilizing the polyhistidine tags present on both MBP and TEV protease with immobilized metal affinity chromatography to separate them from the protein of interest following digestion with TEV Protease.
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