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 Recktenwald, PhD
IBEX Technologies, Inc.
5485 rue Pare
Montreal, Quebec, H4P 1P7
Fax : (514) 344-8827
email: achim at ibex.ca
The usual disclaimers apply.
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