It is entirely possible to elute from a MonoQ matrix with a change in
pH. It will not ruin the column between pH 2-12. Phosphate or Tris or
a mix of buffers would be fine. It would be difficult, except for a
monoprotic buffer in a narrow range, to achieve a truly linear gradient
of pH. I don't see why this really matters. As long as you find
conditions at which the protein binds, and then conditions at which the
protein but not everything else in the mixture elutes, you win. It is
also common to elute from a MonoQ with salt, or with a mix of salt and
pH gradient. Nadir is right below that for a quaternary amine resin,
you are titrating relative to the pI of the protein, since the matrix
will be positively charged at all pH values. That means you need to
bind at a pH higher than the pI (so that the protein is net negative)
and titrate toward the pI (lower pH) to reduce the affinity for the
positive resin. See:
http://www5.gelifesciences.com/aptrix/upp00919.nsf/Content/71857706466D1AB8C1256EB40041805D/$file/11000421AA.pdf
Tom
Nadir T. Mrabet wrote:
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
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Pr. Nadir T. Mrabet
Cellular & Molecular Biochemistry
INSERM U-724
Nancy University, School of Medicine
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France
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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
----------------------------------------------------------------------------
Matthew LH Chu
PhD Student
School of Pharmacy and Pharmaceutical Sciences
University of Manchester
----------------------------------------------------------------------------
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Assistant Professor
Department of Chemistry
& Department of Biochemistry
The Ohio State University
1043 Evans Laboratory
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