Dr. Stefano, 1) I would add FAD in all buffers after lysis,even after GF you could perform dialysis with buffer supplemented with FAD. 2)for the second question I found this paper which could be helpful: Large-scale preparation and reconstitution of apo-flavoproteins with special reference to butyryl-CoA dehydrogenase from Megasphaera elsdenii. Hydrophobic-interaction chromatography. Good luck, Mirella
Sent from my iPhone On 13 Mar 2014, at 22:40, "Benini Stefano (P)" <[email protected]<mailto:[email protected]>> wrote: Dear All (those dealing with wetlab stuff..), While purifying a FAD containing protein we lose part of the FAD (on the gel filtration we clearly see two bands corresponding to holoprotein and free FAD). We obtain crystals but diffracting to only about 4 A despite their beautiful look. Our hypothesis is that the crystals contain a population of molecules with and without FAD (?). The questions are: 1) how to keep FAD bound to the protein during purification and crystallization? 2) how to completely remove FAD from the protein? Thank you very much for any help provided! Best regards Stefano (part-time wetlab person) Dr Stefano Benini, Ph.D. Assistant Professor First International workshop: "Molecular Basis of Fire Blight", Bolzano 15.10.2014 Laboratory homepage: http://pro.unibz.it/staff2/sbenini/B2Cl.htm Personal homepage http://pro.unibz.it/staff2/sbenini/ "I don't like anything that's fake and I hate pretenders!" ********************************************* Bioorganic chemistry and Bio-Crystallography laboratory (B2Cl) Faculty of Science and Technology Free University of Bolzano Piazza Università, 5 39100 Bolzano, Italy Office (room K2.14): +39 0471 017128 Laboratory (room E.021): +39 0471 017910 Fax: +39 0471 017009 ******************************************** "ogni giorno in più è un giorno in meno."
