Why is there is so much target protein in the column flow through?

If the yield of target protein in the eluate is satisfactory, it is likely that the target protein is well expressed and the capacity of the beads has been reached. If the eluate yield is also low, it is possible that the beads were not mixed well with the crude lysate. It’s recommended to perform the binding step using end over end mixing or by using a benchtop shaker at 850 rpm to keep beads in suspension. If beads settle to the side or bottom of the well or tube throughout the isolation, less efficient binding of the target will occur. In addition, optional placement of the His-tag is protein specific. High levels of target protein in the flow through may be a result of the His-tag being sequestered within a fold of the protein thus making it unable to interact with the immobilized Nickel ion responsible for the affinity. Carrying out the purification under denaturing conditions often resolves this problem. Another option is to move the His-tag to the opposite terminus of the protein to make it more accessible.

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