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: [EMAIL PROTECTED]
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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
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Matthew LH Chu
PhD Student
School of Pharmacy and Pharmaceutical Sciences
University of Manchester
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