Michael,
Well, why do you need to titrate the exchanger rather then the proteins
themselves?
MonoQ is a lot simpler to adjust pHwise, as with DEAE you actually
titrate both the matrix and the proteins.
Recommended buffers to use are Goods' (pKa from 8 to 6.15 at 20 °C) +
acetic acid (pKa 4.76).
An equimolar (50mM) mixture of these with Buffer A titrated to 8.0 and
Buffer titrated to 4.0 has been
shown (in my hands) to yield a very linear gradient (must not be too
steep, though).
Matthew's question does not seem to concern chromatofocusing.
Hth,
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]
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.
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
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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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