How can an on-column cleavage reaction be carried out if the target protein has optimal activity at low salt?
The composition of the Cleavage Buffer is not critical for the cleavage reaction and can be made similar to the final storage buffer for the target protein. Wash the column extensively (at least 20 column volumes) with high salt Column Buffer (this removes proteins that may bind to chitin by ionic or other types of nonspecific interactions). Wash the column with at least 3 volumes of the low salt Cleavage Buffer (without DTT or 2-mercaptoethanol) and then flush with 3-5 volumes of DTT-containing Cleavage Buffer. This ensures that the target protein is eluted in the specified buffer.
