Protein recovery from columns

tivol at tethys.ph.albany.edu tivol at tethys.ph.albany.edu
Fri Jun 17 10:29:56 EST 1994


Dear Alex,
	The cost of a spoonful of packing material depends, of course, on the
material.  I'll date myself by saying that I have done this procedure with
things like Sephadex and DEAE.  These are very inexpensive.  The packing
material for HPLC columns is much more, but this is due primarily to the
small and uniform size required for HPLC.  If you can find some material
which is chemically identical, you can almost certainly use it instead of the
"official" column packing.  If, however, the column packing has a particular
internal geometry, e.g. a zeolite, make sure you substitute a material with
the same pore size, etc.
	When I said "shake", I was not being literal.  Swirling the mix of
protein in buffer and packing material is more like what I actually did.  You
can just let the mix sit in the beaker without any form of agitation if you
want; the experiment just takes longer that way.  In fact, ideally, you want
to subject the protein-packing mix to the conditions it will see on the col-
umn.  One way this procedure can fail is if the specific volume of an aggre-
gate of your protein is much less than the sum of specific volumes of the
unaggregated form.  In this case, such forces as electrostatic, solvation,
etc. can overcome the PV energy at low (atmospheric) pressure, but not at
high pressure.  The mix in the beaker will not aggregate, but when subjected
to HPLC, it will.  The trick then is to find conditions which increase the
net charge or solvent interaction to overcome the higher PV energy and which
do *not* denature the protein--a non-trivial task.  Good luck.

					Yours,

					Bill Tivol



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