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 Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij 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 >> uday
