Troubleshoot Chocked Ni-NTA Column - (Apr/08/2006 )
Hi everybody,
I was using Ni-NTA column for His-tagged protein and everything was going well. Generally before addding anything to the column I used to filter solutions using 0.45u filters. Today I forgot to filter the protein supernatant and added it whole to the column as such. The flow rate dropped remarkably. Realising my mistake after an hour or so, I took out whatever was left on the top and filtered it but I think I am late. The flow is slow!! 
Please suggest me how to get the flow back of this choked Ni-NTA column.
Waiting eagerly for your response.
Best Regards,
Amit Kumar
-Bio Tyro-
you'd better use urea to wash the column
-solidoil-
QUOTE (Bio Tyro @ Apr 8 2006, 07:39 PM)
Hi everybody,
I was using Ni-NTA column for His-tagged protein and everything was going well. Generally before addding anything to the column I used to filter solutions using 0.45u filters. Today I forgot to filter the protein supernatant and added it whole to the column as such. The flow rate dropped remarkably. Realising my mistake after an hour or so, I took out whatever was left on the top and filtered it but I think I am late. The flow is slow!!
Please suggest me how to get the flow back of this choked Ni-NTA column.
Waiting eagerly for your response.
Best Regards,
Amit Kumar
I was using Ni-NTA column for His-tagged protein and everything was going well. Generally before addding anything to the column I used to filter solutions using 0.45u filters. Today I forgot to filter the protein supernatant and added it whole to the column as such. The flow rate dropped remarkably. Realising my mistake after an hour or so, I took out whatever was left on the top and filtered it but I think I am late. The flow is slow!!
Please suggest me how to get the flow back of this choked Ni-NTA column.
Waiting eagerly for your response.
Best Regards,
Amit Kumar
Try a reverse flow with NaCl and NaOH after stripping the column and then recharge it- you can find the instructions at the GE Healthcare-website (search for your column, then watch for the "manuals"-link), under the keyword "cleaning in place". Works fine with my columns :-)
good luck!
-Susannah-