Dear All
On similar lines any suggestions and tips on expression and
purification of the proteins containing both FAD and NADPH would be really
helpful.
I recently tried to express and purify a protein containing these two
ligands, but failed (aggregation of protein).
my initial questions are
K] On Behalf Of Benini
Stefano (P)
Sent: 13 March, 2014 18:40
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 clearl
Stefano,
Before you address the problem, you need to ask yourself a couple of things.
You say that "on the gel filtration we clearly see two bands corresponding to
holoprotein and free FAD." That is not too odd, but have you ask the question
is all the protein good protein.
Is this an enzy
, 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
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 but
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
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
o
(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 cl
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 hypot
