Hi I am trying to crystallize a Fe-S cluster containing protein which is sensitive to oxygen present in the air. In the presence of air due to 'fenton reaction' the Fe-S breaks to give heterogeneous population of different Fe-S cluster and free Fe species. I also faced lots of difficulties in the purification of this protein with different tags as it always carries contaminants with it. However mass spectrometry have suggested some of them to be our protein of interest which was just an artifact of the SDS PAGE which appeared due to presence of iron in the protein.
My questions are: 1> Why even after boiling the sample in loading buffer i see multiple bands of my protein of interest. Has anybody seen and observed this profile for any metalloproteins previously ? 2> We don't have an anaerobic crystallization set up then what should be best way to avoid Fe toxicity before crystallization trials. Has anybody successfully crystallized any Fe-S cluster containing protein by chelating it off by EDTA and Ferricyanide. I used it once but I still see some residual brown color on the base of amicon filter during concentration. 3> Are there any other better way to chelate Fe-S from the proteins ? Your suggestions will be highly helpful for me. Regards Kahkashan
