Hi Nadir, No I didn't and I am not concerning CHROMATOFOCUSING, and you are right, I have been thinking of using both pH and salt gradients to refine the separation. Thanks!
Matt 2008/6/25 Nadir T. Mrabet <[EMAIL PROTECTED]>: > John, > > You can adjust your ionic strength not only with NaCl/KCl/etc but also by > increasing the concentration of the components > in your buffer mixture. If you do it right, I can assure you can get a > linear pH gradient. > And, as mentioned earlier by Tom, you can use both pH and salt gradients to > refine your separation, even batchwise (no linear > gradient required... if it works). > > The problem working with pH gradients is re-equilibrating the resin, since > what you are actually doing is titrate a supposedly > high-capacity buffer with another high-capacity buffer. Therefore, patience > is required and sufficient volumes of buffer A. > > Again, I ask the question: Did Matthew ever mentioned before he intended to > perform a CHROMATOFOCUSING > experiment? > > Nadir > > -- > > Pr. Nadir T. Mrabet > Cellular & Molecular Biochemistry > INSERM U-724 > Nancy University, School of Medicine > 9, Avenue de la Foret de Haye, BP 184 > 54505 Vandoeuvre-les-Nancy Cedex > France > Phone: +33 (0)3.83.68.32.73 > Fax: +33 (0)3.83.68.32.79 > E-mail: [EMAIL PROTECTED] > > > > John A. Newitt wrote: > >> 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 >> > -- ---------------------------------------------------------------------------- Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester ----------------------------------------------------------------------------
