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
