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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. Andreas has pointed you in the general direction for chromatofocusing, but there is a "poor man's" way to do it. We use this method a lot, and the key is using a weak ion exchanger (like DEAE or CM) and a mix of buffers with pKas that span the titration range you want to exploit. Remember, you actually want to titrate the resin with the buffer: as the pH shifts away from the pKa of one buffer component, it moves into the buffering range of the other. If you do it correctly, you get a nice, flatter titration curve from the resin, which spreads out the release of the proteins. We have used a mixture of Tris and Bis-Tris-Propane with a HiTrap-DEAE or Sepharose- DEAE FF columns. Hope this helps, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: [log in to unmask] **************************************************************** On Jun 24, 2008, at 12:53 PM, Matthew Chu wrote: > Dear All, > > Sorry for off-topic question. Does anyone have any experience in > purifying protein using pH gradient in Mono Q column? > > I have been googling for a whole day, only one paper was found to > mention performing pH gradient in Mono Q, but in a mixture of amine > buffering species, which is a bit too complicated (J. Chromatogr. A > 1164 (2007) 181 - 188. Can Tris-Cl/Tris-base or phosphate buffer > give a linear pH gradient from pH 8.0 to 4.0? Is it usual to > perform pH gradient in Mono Q as I don't want to ruin my Mono Q > column... > > Any suggestions are welcome. Thanks in advance! > > Kind regards, > Matt > > > ---------------------------------------------------------------------- > ------ > Matthew LH Chu > PhD Student > School of Pharmacy and Pharmaceutical Sciences > University of Manchester > ---------------------------------------------------------------------- > ------