Hi Matt,
In addition to the other helpful comments, I have also found that if
you are using reducing agent, this complicates
issues furthers due to metal complexation, I suppose. If you do need
a reducing agent
for your protein, I would suggest adding TCEP instead of BME or DTT,
as this
has changed solution color the least for me. Just make sure you add
the TCEP and buffer,
pH the solution, and then add the metal ion of interest as everyone
has suggested, since TCEP is acidic.
A protein I work with is active at pH's of around 8, and TCEP in my
buffers (Bicine or Tris) seems to help slow
precipitation, but at 5 mM Mn2+ at that pH there will be precipitation
after a few days no matter I guess...
Good luck!
-Dan
On Apr 6, 2009, at 2:14 PM, Matthew Alan Bratkowski wrote:
Hi.
Does anyone have experience using solutions containing manganese as
crystallization buffers (buffers, not screening well solutions)? The
protein that I am working requires manganese for activity, and I
have read
reports of related proteins crystallizing in manganese buffers. I
made a
buffer containing 3 mM manganese, that initially had a black color
then
turned to deep purple, and later almost clear. After a few weeks, the
buffer turned an orange color and contains dark manganese syrup on the
bottom. Does anyone know how to prepare a manganese buffer that is
stable
enough to be used as a protein buffer for crystallization screens?
Thanks,
Matt
