washing buffer retained in Ni-NTA column after centrifugation - (Oct/05/2007 )
hi all,
i having problem using Ni-NTA spin column from qiagen. i followed the protocol to purify protein under denaturing condition. however, during the washing step, the washing buffer tend to retain in the column. it always took me more than 30 minutes instead the 2 minutes (manufacturer protocol) for single washing step. what can i do to prevent this problem?
Thanz.
-angelet-
QUOTE (angelet @ Oct 5 2007, 10:41 AM)
hi all,
i having problem using Ni-NTA spin column from qiagen. i followed the protocol to purify protein under denaturing condition. however, during the washing step, the washing buffer tend to retain in the column. it always took me more than 30 minutes instead the 2 minutes (manufacturer protocol) for single washing step. what can i do to prevent this problem?
Thanz.
i having problem using Ni-NTA spin column from qiagen. i followed the protocol to purify protein under denaturing condition. however, during the washing step, the washing buffer tend to retain in the column. it always took me more than 30 minutes instead the 2 minutes (manufacturer protocol) for single washing step. what can i do to prevent this problem?
Thanz.
Did you filter both, the washing buffer and your solution which was loaded on the column? Best to use a 0.45 or 0.2 micrometer PES filter.
-Dukes-
What speed are you using for spinning down the washing buffer? It might be the centrifugation.
And yea.. some columns are abit dodgy.
-timjim-
QUOTE (timjim @ Oct 6 2007, 11:25 AM)
What speed are you using for spinning down the washing buffer? It might be the centrifugation.
And yea.. some columns are abit dodgy.
And yea.. some columns are abit dodgy.
i used 700xg, which is the highest speed should be used for this purification.
-angelet-
QUOTE (angelet @ Oct 5 2007, 04:41 PM)
hi all,
i having problem using Ni-NTA spin column from qiagen. i followed the protocol to purify protein under denaturing condition. however, during the washing step, the washing buffer tend to retain in the column. it always took me more than 30 minutes instead the 2 minutes (manufacturer protocol) for single washing step. what can i do to prevent this problem?
Thanz.
i having problem using Ni-NTA spin column from qiagen. i followed the protocol to purify protein under denaturing condition. however, during the washing step, the washing buffer tend to retain in the column. it always took me more than 30 minutes instead the 2 minutes (manufacturer protocol) for single washing step. what can i do to prevent this problem?
Thanz.
oh... i dint...
i think i should try...
thanz...
-angelet-
Just a question, does it really matter to filter the washing buffer? Oh great.. protein purfication is just tedious.
-timjim-