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weak binding to ni-nta column under denaturing conditions

weak binding to ni-nta column under denaturing conditions - Protein Forum

weak binding to ni-nta column under denaturing conditions - Protein Forum

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Old 07-21-2005, 09:09 AM
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Default weak binding to ni-nta column under denaturing conditions

I am expressing a protein with c-terminal his-tag in bl21(de3) cells as
inclusion bodies. My problem is that even under denaturating conditions
binding of the protein to the ni-nta qiagene column is very low. In
fact, no binding occures if i follow a denaturating protocol (changing
buffer pH values). Some of the protein binds to the column if native
imidazole (10, 20, 250) protocol supplemented with 8M urea is followed,
but still majority of protein is eluted in wash fraction. To avoid the
column flow problem i did several mini-preps with prolonged incubations
and stirring options but the result remains the same. Increasing the
total salt concentration up to 2M did not help, neither did addition of
2-mercaptoethanol. I cant afford anti-his antibodies and will try
adding more protease inhibitors. Any suggestions or experience with
changing the his-tag orientation or maybe even adding a second his-tag?


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binding , column , conditions , denaturing , ninta , weak

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