If the third line from the left of marker is your purified protein sample, then your work of washing the impurity protein is not enough. I suggest you do a small system experiment to optimize the conditions for washing the protein, and then continue the experiment.

如果从marker左数第三道是你的纯化后蛋白样品，那么你洗杂蛋白的工作是不够的。建议你先做小体系的实验优化下洗杂蛋白的条件，再继续这个实验

Hello Zhao. Thank your for your kind response.

I have attached here the overexpression profile of that particular protein not the fraction of purified protein.

When I purified the protein, I have got a faint band of this protein (total 2mg protein from 7liter culture ) with less impurity.

Hope you get your answer.

Thanks.

Riya Karan, please check the flow-through if you are losing some protein. This may happen because of improper binding of recombinant proteins to the affinity column. It has been observed that sometimes affinity tags (e.g: 6X his-tag) fold into the core of the protein which hampers the binding to the resin. I would rather suggest to check your flow-through.

Hi, you may try following:

1. Adding PMSF in the lysis buffer

2. Use freshly recharged column

3. As Shakilur Rahman suggested, check the flow through for the inefficient binding of protein to the column.

4. If using FPLC system, try passing the sample at low rate (0.1 ml/min) through the column.

Also, a SDS-PAGE image with the over expressed protein, flow through, column washes, and the eluted protein will give more insight into why this is happening.

Metallo beta-lactamases sometimes give lesser yield due to insufficient zinc in the buffers. In that case, dialyzing the sample against zinc containing buffer helps.

Thank you Shakilur Rahman for your suggestion.

I have already checked the flow through, there was almost no protein in the flow through. The protein is bound properly in the colume.

Thank you again.

Thank you Diamond Jain for your kind response.

I had done the necessary optimization that you suggested, but I could not get a satisfactory result.

Thank you again.