Ion Exchange - (Jan/27/2009 )

I am running an ion exchange column with DEAE Sepharose FF and I am separating ferritin in 25mM TRIS with a linear gradient from 0 to 0.400M NaCl in 25mM TRIS. This is my first time using an IEX column and had a few questions.
First after i ran the column, and eluted my protein there was a faint yellow band at the top of the gel, i presume left overs of the sample, but i attempted to force it off the column with 2M NaCl and it still would not budge. Does anyone have any incite on this situation.
Secondly, the sample that i am injecting is 0.200ml and when i collect all of the solution that gave a signal on my UV detector i ended up with almost 200ml of solution, there was also visual confirmation that it was in fact ferritin coming off of the column since it was yellow. Is this a normal amount for a sample of that size to increase to that kind of volume?
Thanks for any suggestions or comments.

Hello

I am not sure if this would help, but worth reading.

I can't comment on the first question you raised......however, if it didn't come out with 2M NaCl, its less likely to interfere with yr further analysis. Can u elute ferritin in somethign else, which may just bring it down?

For the second question, its a trade off. WhenI purify my protein on ion exchange, I surely purify it, but it gets diluted out. So whereever i am looking for concentration, i don't look for purity. What i mean is that i load samples which are relatively pure and elute them out using a running gradient so that everything comes out in a low volume but surely at a higher concentration.

Best
TC

Can i ask how large of a column you are running for your IEX?

this handbook from ge healthcare may help.