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Add the clear sample containing target GST-tagged protein onto the equilibrated gravity column, keep the sample in column for at least 2 min, ensure that the sample fully binds with the resin, and collect the flowthrough with a flow-rate of 0.5 - 1 mL per minute, then add the flowthrough onto the column again, repeat the previous operation, this can increase the binding efficiency

Pereira JPC, Verheijen PJT, Straathof AJJ (2016) Growth inhibition of S