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
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
Dear Stefano,
Here few thoughts:
You should calculate the amount of holo/apo protein that you get after the
column. A ratio 280/450 between 9-12 it will suggest that your protein is
almost completely in the holo-form. So, I will be worried about the peak of
flavin only if this ratio become
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
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
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
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
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
