separation of two negatively charged proteins.... - ion exchage chromatography??? (Jul/02/2006 )

yes im still trying to separate native and degraded forms of my expressed protein..

i calculated charge of the native protein and it is -15 at ph 8.8....while cleaved form of the same protein is -20 ....my professor suggested i separate them on affinity chromatography according to charge playing with salt concentrations... .....i can't understand it...did he mean ion exchange chromatography? can it be used to separate 2 peptides both negatively charged? maybe i change pH so that one becomes positively charged??? ....please tell me what you think about this....

yes im still trying to separate native and degraded forms of my expressed protein..

i calculated charge of the native protein and it is -15 at ph 8.8....while cleaved form of the same protein is -20 ....my professor suggested i separate them on affinity chromatography according to charge playing with salt concentrations... .....i can't understand it...did he mean ion exchange chromatography? can it be used to separate 2 peptides both negatively charged? maybe i change pH so that one becomes positively charged??? ....please tell me what you think about this....

Either process should work in theory. Unfortunately, reality has a nasty way of totally disregarding theory.
Depending on how your affinity column works, you might be able to separate the two forms if the degraded from doesn't bind well. If it does bind, salt may very well separate the two, or else try pH or whatever ligand you use to bind to the column in the first place (you might get lucky and be able to elute the gdegraded form with a low level of ligand, and then elute the folded form with a high level...).
If you go to ion exchange, your options open up a bit. The degraded form of your protein will have a different surface charge to your active form. Ion exchange chromatography should work ok with such a difference of charge. A point of warning: the sufrface charge is clearly very much dependent on structure, so you might have a significant difference between the charge you have calculated and what is happening in real life...
As a part of your ion exchange process, don't forget that you can use either strong or weak exchangers. The benefit of a weak exchanger is that you can use not only salt concentration, but also pH changes to separate proteins.

Good luck. It seems that you are soooo close to nailing this problem.