Help:how to save my Ni column - (Nov/25/2004 )

when I use my Ni column to purify my his-tag protein that I added 1mM DTT,I found that the colour of the column has changed !!It became brown!!
what can I do to save my Ni column?
I just used EDTA and 0.1M NaOH to wash the column,but it doesn't work.
maybe somebody can help me ,otherwise I'll be upbraided by my boss.

Check here:

http://micro.nwfsc.noaa.gov/protocols/meth...10.01.2191.html

The brown colour shouldn't affect the performance of the column.

When using EDTA or other chelating agent you are loosing the Ni chelated by the NTA. If you loose Ni on the resin, it's color goes brown.
And of course if you loose Ni you loose protein loading capacity.
Your purification isn't optimal and you will obtain bad protein levels....

You ought to charge your colum with Ni2+ (see Qiaexpressionist PDF)

Download this PDF document :
http://www1.qiagen.com/HB/QIAexpressionist

You will find lots of information about NiNTA purification system. You have also a table with compatibility reagent. If you can limit the use of chelating agent for your purification it's better for sure...

Good luck
David.

thank for all of you.