Hi Stefano,
On top of all that has been suggested you should also be aware of the effect of
pH and buffer composition on the apo-holoenzyme equilibrium during purification
and crystallization.
Boaz
Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel
E-mail: [email protected]
Phone: 972-8-647-2220 Skype: boaz.shaanan
Fax: 972-8-647-2992 or 972-8-646-1710
________________________________________
From: CCP4 bulletin board [[email protected]] on behalf of Benini Stefano
(P) [[email protected]]
Sent: Friday, March 14, 2014 12:40 AM
To: [email protected]
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:
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!"
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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
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