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My protein doesn't bind to the Ni-NTA column - (Nov/07/2012 )

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The large band appears the right size for your fusion protein.  Is it?  If so, it expressed very well and would definitely overload a 1ml column but your yields should still be better.  I would first try removing the gradient and eluting all at once with a high concentration (as mdfenko suggested) and include a flow thru sample on your gel.  It is unlikely that you are losing your sample (binding to the column and not eluting) but evaluating the flow thru can be helpful.  If your expression level is as high as your lysate seems, then your yields should be high and there are no flags for me in your protocol.  So, I'd probably try denaturing some lysate to see if the tag is somehow hindered reducing the amount that can bind....

-Missle-

The large band appears the right size for your fusion protein.  Is it?  If so, it expressed very well and would definitely overload a 1ml column but your yields should still be better.  I would first try removing the gradient and eluting all at once with a high concentration (as mdfenko suggested) and include a flow thru sample on your gel.  It is unlikely that you are losing your sample (binding to the column and not eluting) but evaluating the flow thru can be helpful.  If your expression level is as high as your lysate seems, then your yields should be high and there are no flags for me in your protocol.  So, I'd probably try denaturing some lysate to see if the tag is somehow hindered reducing the amount that can bind....

Thank you for your suggestion! I would try it tomorrow. Maybe it would be useful. Then i will tell you the results. Thank you!

-yingquliang-
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