Hydroxyapatite columns

Achim Recktenwald reckteac at ibex.ca
Tue Apr 22 16:20:19 EST 1997


Thomas Seib wrote:
> 
> In article <335BA20C.57AC at nott.ac.uk>, Simon.Dawson at nott.ac.uk wrote:
> 
> > Hello all,
> >
> > I'm attempting to purify a protein complex from an insect muscle and as
> > a third stage of purification I'll be using an hydroxyapatite column. As
> > a test exercise I've bound lysozyme to the column and eluted, that
> > worked fine. But, again as a test prior to loading my protein complex of
> > interest, I've tried to load an extract of total soluble muscle proteins
> > from the insect I'm working on. I found that virtually none, if any, of
> > the muscle protein has bound.  Why not?  I'd assumed that most, or at
> > least some proteins from muscle would bind, but looking at the FPLC
> > protein concentration trace everything seems to come straight through
> > the column.
> > The column buffer I've used is a potassium phosphate buffer of pH 7.6
> > (I've also tried pH 6.8) containing 1mM beta mercaptoethanol and 10%
> > glycerol. I've used an elution gradient of 20-500mM phosphate. The total
> > protein sample buffer would also contain small amounts of Tris (1mM),
> > DTT (0.01mM), ATP (0.01mM) and MgCl2 (0.05mM).
> >
> > Help please!!
> >
> > I don't really want to load my precious protein  complex until I'm happy
> > with the column (even though I know that I can always collect the
> > unbound material).
> >
> > Cheers,
> >
> > Richard.
> >
> > --
> >
> > Richard Hastings       TEL:+44 (0)115 9249924 ex. 44787
> > Dept. of Biochemistry  FAX:+44 (0)115 9422225
> > Queens Medical Centre  Email:Simon.Dawson at .nott.ac.uk
> > Clifton Boulevard
> > http://www.ccc.nottingham.ac.uk/~mbzspd/Simon.html
> > Nottingham
> > U.K.                   "Back off man, I'm a scientist!" - Bill Murray.
> 
> Hello Richard,
> 
> 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.
> 
> Sincerely, Matthias
> 
> --
> Thomas Seib
> Kantstr. 12
> 66292 Riegelsberg


You also have to consider the ionic strength of your sample. If it is
too high the proteins will not bind.

Achim
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Achim Recktenwald, PhD
IBEX Technologies, Inc.
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