Hi Stefano,
Wouldn't you be better off going the other way around, that is supplementing your protein solution with FAD prior to setting up the crystallization trials? It sounds as if the FAD is quite labile in your case, but it could well stabilize the protein and hence give rise to better diffracting crystals. Unless of course you have good reason to believe that the apoprotein is more stable, in which case dialysis could be the way to go in order to get all (??) the FAD out.
My 2p thoughts.
Good luck,
Boaz
Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel
E-mail: [log in to unmask]
Phone: 972-8-647-2220 Skype: boaz.shaanan
Fax: 972-8-647-2992 or 972-8-646-1710
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From: CCP4 bulletin board [[log in to unmask]] on behalf of Benini Stefano (P) [[log in to unmask]]
Sent: Friday, March 14, 2014 12:40 AM
To: [log in to unmask]
Subject: [ccp4bb] off-topic: protein losing FAD during purification
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:
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