Thank you for all your advices. I usually employ salt gradient in MonoQ for my proteins, but I just wonder if we can use pH gradient in the same column and see if it can improve the separation.
I know the condition that my protein binds to Mono Q, so I will try to titrate toward its pI to reduce the affinity of the column. I am going to try Tris / phosphate buffer first, will let you guys know the results. Really appreciate for all your advices again. Matt 2008/6/25 John A. Newitt <[EMAIL PROTECTED]>: > At 1:50 PM -0400 6/24/08, R.M. Garavito wrote: > > Matthew, >> >> You're not going to ruin your column, but you won't get great performance >> either. Elution by pH change is a very common method, but getting a really >> linear pH gradient is very hard. The Mono Q matrix is a strong anion >> exchanger, meaning that it is insensitive to pH changes, i.e., you can't >> titrate it smoothly with acid or base. DEAE resins, which are weak anion >> exchangers, have a nice pH titration curve and lend themselves better to >> elution by pH change. This is the reason chromatofocusing is not a commonly >> used method, and its expensive. >> > > There is a company the sells a proprietary buffer system and gradient > programming calculator to create a stable pH gradient for separation on a > MonoQ column or other strong ion exchanger. > > <http://www.cryobiophysica.com/> > > My problem with pH gradient techniques is that they don't work very well > unless your protein is happy in low ionic strength buffers, which is almost > never the case with my projects. This company now claims that it can create > the pH gradient with NaCl present, but I haven't tried this yet. > > - John > -- > <http://xri.net/=john.newitt> > -- ---------------------------------------------------------------------------- Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester ----------------------------------------------------------------------------
