And include FAD at a few uM in your column buffers. Although if you are getting
two separate sharp peaks for protein and FAD, it doesn't sound like it is
bleeding
off during chromatography but rather already dissociated in the material you
apply
to the column. Take a spectrum of the material in the peak you suspect of being
FAD-
Free FAD has a huge peak at 260 and smaller peaks around 375 and 450.
Boaz Shaanan wrote:
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: bshaa...@bgu.ac.il
Phone: 972-8-647-2220 Skype: boaz.shaanan
Fax: 972-8-647-2992 or 972-8-646-1710
________________________________________
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Benini Stefano
(P) [stefano.ben...@unibz.it]
Sent: Friday, March 14, 2014 12:40 AM
To: CCP4BB@JISCMAIL.AC.UK
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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Faculty of Science and Technology
Free University of Bolzano
Piazza Università, 5
39100 Bolzano, Italy
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