just out of curiosity, was one Se enough in this case to solve the structure of your 0.2 kD protein by MAD?
Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
On 17 Apr 2012, at 18:13, James Holton wrote:
> 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