Hydroxyapatite columns

Zophonias O. Jonsson zjons at vetbio.unizh.ch
Tue Apr 22 16:17:13 EST 1997

In article <hgtsei-2204971417500001 at ukmhg03.med-hg.uni-sb.de>,
hgtsei at med-rz.uni-sb.de (Thomas Seib) wrote:

> a very simple suggestion, maybe, but did you desalt the total muscle
> proteins before loading onto the column? I think of Ca2+-Ions in the 
> extract that could be masking PO42- binding sites in the column.

This may or may not work, depending on the pI of the protein (complex).
HAP has rather strange characteristics.  If you are purifying a basic
protein the above suggestion is good, but if your protein is acidic it
will bind tighter in high salt.  I regularly use ceramic HAP (which by the
way is much more agreeable to work with than regular HAP) to purify a
protein with pI= 4.5.  It binds tightly in 2M NaCl and can be washed with
2M MgCl2 but is eluted at mM concentrations of phosphate in 50 mM NaCl.

The problem may actually be the trace of MgCl2 in the buffer described in
the original post since MgCl2 is good at eluting basic proteins from
phosphate buffered HAP. Dialysis or desalting would fix that.

For some HAP theory and good suggestions take a look at:

Gorbunoff M.J. (1985) Methods in Enzymology, vol 182, 329-339

Good luck


Zophonias O. Jonsson
Institut fur Veterinarbiochemie               Tel: (41-1)-257-54-75
Universitat Zurich-Irchel                     Fax: (41-1)-362-05-01
Winterthurerstrasse 190
CH-8057 Zurich

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