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
ha
Hi Matthew,
Two anecdotes:
For one protein we had protein in pH7 with 10 mM Mn2+ and just set up
crystal trials as normal. Although at higher pH excess Mn precipitated
as MnO2, we still had Mn (2+, presumably) in the active.
For another protein, we grew apo crystals at pH8 and then gradually
I have used up to 20 mM MnCl2 in acidic buffers without visible changes in the
color of the solution. Unfortunately it changes quite dramatic above pH of 7-
Probably due to the formation of Mn(OH)2, which Arthur already mentioned. It
might be further oxidized to a Mn3+ species.
During a purifi
We regularly use 1-2 mM Mn2+ in xtal set-ups. The problem we've
encountered is formation of the insoluble, brown-colored Mn(OH)2 in
more basic solutions, but we've also found that the Good buffers
(HEPES, EPPS, etc.) are less prone to the formation of the
precipitate, while Tris seems to p
Dear Matt,
I use manganese for all my kinase buffers. My holobuffer contains 2mM
manganese at pH 7.0. The key was to add manganese after you pHed the
buffer with NaOH. I never used any basic buffer and it could be issue
if your buffer is too basic. Below is the recipe for my holobuffer. I
Presumably you want Mn2+ , but you need to specify.
If so, you need to make it up fresh, and keep it at a pH below 7 if at
all possible, as it oxidizes readily.
Bis-tris will weakly chelate it and slow the oxidation process.
On Apr 6, 2009, at 11:14 AM, Matthew Alan Bratkowski wrote:
Hi.
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
