protein purification - (Jan/09/2008 )

hi guys,

i am doing fusion protein purification using affinity chromotography and aion exchange chromotography. recently i always met the same probem. after loading the sample to anion exchange, i couldn't get my protein and after using elution buffer, the objective protein was eluted together with other proteins.

the first time i met this problem is because something wrong with the pH of binding buffer and the equlibrate time is not enough, but later there is no problem with the buffer and the sample loaded is also good. i really don't know what is the problem.

ion exchange is relatively non-specific. if you do a batch elution then you will get more than one protein eluting (assuming you put a protein mixture on the exchanger).

you can try a gradient elution.

maybe salt concentration is too high in your sample, or pH is not right?

thanks a lot for reply! first i used the binding buffer to elute the objective protein, then used high salt concentration elution buffer to get rid of other proteins. i did several times using this method before and it worked well. about the sample salt concentration, it also worked well before. i was wondering if there is any problem about my column, this time i used 40mL column, and before that i used 20ml.

tell us more about how did you do the experiment.