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Nickel Column Partially Drying Out

Nickel Column Partially Drying Out - Chromatography Forum

Nickel Column Partially Drying Out - Discuss Chromatography techniques in this forum. Includes a HPLC forum, and other "liquid phase" separation techniques.


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Old 11-16-2012, 05:26 AM
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Default Nickel Column Partially Drying Out



I am completely new to the area of protein purification, so I apologize if this is a basic question. I know that as a general rule, it is never good to let any type of column run dry at any time during a purification. This is only my second time attempting to purify a protein with a modified thioredoxin (Invitrogen, His-path thiofusion expression system) as the tag, which binds nickel. Thus, I am using the Invitrogen Probond resin to purify my protein and unfortunately during my last fraction, I ran out of eluting buffer and could not stop the flow before the buffer had gone a little below the top of the resin bed. I added some binding buffer after I had calmed myself down a little (I was afraid that I just ruined the column).

My question is: is the entire column ruined, and do I have to pour a new column? I saved the column under the assumption that it was still usable. My reasoning was that at most, the top 10-20% of the column may have dried out during that time (this is just a wild guess since there is no way of me knowing for sure), which means the majority of the column should still be pretty good.
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