jjmirujo at unav.es
Sat Mar 4 05:16:07 EST 2000
"Lars H. Østergaard" wrote:
> Hi everybody
> Any ideas on how to monomerize a dimer (hydrophobic interaction) - and
> then afterwards
> being able to form an active dimer again? Would a drop in
> salt-concentration in the buffer
> do the job? Or a mild detergent? Any suggestion would be gratefully
In fact, it depends on the strength of the interaction. Try to prepare
your protein in a solution containing different concentrations (5%, 10%,
15%, 20%...) of an organic solvent (methanol, ethanol, acetonitrile...) in
a suitable buffer at 0-4 oC (dilute 1:1 or dialyze against this buffer).
Remember that the absolute concentration of the protein affects to the
monomer-dimer equilibrium, so test several concentrations of your protein.
Measure the relative amount of monomer-dimer by gel filtration
chromatography (a high performance column will be needed to separate
them). You can also follow the process by measuring enzyme activity (a
100-fold dilution may be necessary to avoid solvent effects). To get the
dimer again, dilute (or dialyze) your protein in a solvent-free buffer.
Adding a antichaotropic salt (try 0,2-2 M NaCl or (NH2)2SO4) will help.
It is advisable not to dilute so much your protein in this step since the
dimer/monomer relation at the equilibrium will be decreased. On the other
hand, if excesive solvent remains after dilution you can expect some
monomer remaining at the end.
Juan J. Martinez Irujo
Departamento de Bioquimica
Universidad de Navarra
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