"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
> appriciated.
>> Thanks
>> Lars
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.
Good luck.
--
Juan J. Martinez Irujo
Departamento de Bioquimica
Universidad de Navarra
Pamplona, Spain
---
