In cases like this, I keep FAD around during all steps starting from cell lysis, purification, dialysis and even crystallization. In one case we added FAD to protein such that the protein: FAD ratio was 1 prior to setting up crystallization trials. Hope that helps. Harkewal
Sent from my iPad > On Mar 13, 2014, at 5:40 PM, "Benini Stefano (P)" <[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."
