3 replies to this topic

[Benita]

Hey all,

theres a new question from me .

Since a while i use GE Healthcare High Trap Affinity Columns (FF). There filled with Nickel beads. Tha last time after 5 purification with the same column the color turns from light blue to brown-grey. So I thought there was to much amount of protein purified with this column and I took a new one. So in this column the colorchange occurs with the first purification?!? So can you give me a hint what could have been happened? I always use the same protocal etc.

The colorchange occurs at the time I load my protein on the column. It lies in DMEM Media (secreted by my HEK cells) and theres just P/S and no FCS in the media? So I don't think there are any reducing agent sin dmem, right?

When the colorchange occurs theres a smaller amount of purified protein in the end than in purifications without change.

Hope you understand my confused thread.

Thank you in advance!

Franzi!

[Denny]

Hi Franz,
I'm not familiar with DMEM media and not sure what P/S and FCS are. But there is something in your media that is causing problems.

Are you adding any DTT to your buffers and are you performing any DNA precipitations prior to applying your protein to the column?
Nickel will turn brown or purple if DTT is too high or if you use too much PEI - polyethyleneimine, if you have to use it, keep it at or below 1% final volume. I have found that using above 2mM DTT causes problems even though GE states up to 5mM is okay.
Check the manufacturers recommendation for compatible buffers and reagents and compare to your protocols.

Edited by Denny, 01 April 2010 - 03:31 PM.

[Benita]

Hey thank you, but theres no reducing agents in it. I tried it again and it works? Don't know maybe the column was "monday column".

[rudzielec]

I used to strip column every time before use and it's usually helpful but my protein was from E.coli. Different mutant was giving different colour on a column....