If SeMet oxidation is an issue then EDTA is your friend. This is
because Met, SeMet and even Cys-Cys are not oxidized directly by O2,
but rather via a trace transition metal intermediate. So, keeping
enough chelator around to soak up any trace metals will dramatically
slow down the oxidation. I can't remember where I first read about
this, but once upon a time when I was working out how to make fmoc-SeMet
(before you could buy it) and assaying the product by HPLC I found that
I could easily see both the single- and double-oxidized forms on the
chromatograms. Adding H2O2 was a good way to make the oxidized species
(positive control), but a solution of SeMet in water at neutral pH and
~0.5 mM EDTA was stable in air for weeks. I discovered quite by
accident that using a steel probe as a scraper was not a good idea.
Lost a whole batch that way. The singly-oxidized SeMet can be converted
back to SeMet with DTT, but the double-oxidized form cannot.
-James Holton
MAD Scientist
On 4/17/2012 4:03 AM, Uday Kumar wrote:
Hi sarah
I believe, you might have used reducing agent in your SeMet-labeled protein
sample.
if avoiding reducing agent is not a problem to your protein (ignoring
Selenium), try to purify/crystallize your protein in the absence of reducing
agent.
if u want to try this use all degassed buffers.
With regards
uday
