Hi Pascal,
this behavior doesn't surprise me.
We had crystals in FC12, and didn't get any phase transitions in any
PEGs from 200 to 20.000, and with or w/o organic solvents such as MPD.
And I can tell you we had a lot of detergent with the purified protein,
concentrating on 50kDa.
With lipids, the phase diagram is changed radically. Have you tried
bicelles? you have specialists next door. The amount of crystal-like
features is quite high. I would suggest the extensive use of a UV source
to distinguish between "like" and real protein crystals.
good luck.
vincent
Le 8/2/13 12:23 AM, Pascal Egea a écrit :
Dear All,
I have a question tailored for the membrane protein and detergent
folks. We are purifying a membrane protein that associates into an
homoligomeric pore and we have been successfully preparing it in two
detergents: FC-12 or a mild lipid. The two Protein Detergent Complexes
look very homogenous by SEC and can be concentrated without protein
loss on membranes with MW cutoffs (100kDal) way larger that the
"expected" their respective free detergent micelles.
Everything looks good so far... until we get to the crystallization
stage. While the PDC in FC12 does not tend to form too much phase
separation, the PDC in the lipid does. This looks a bit odd to me
since these lipid micelles are supposed to be a bit smaller than the
FC-12 micelles. We are working at twice the CMC and besides lowering
the detergent concentration, I am a bit perplex about what I am
observing. Intuitively I would have expected to observe the reverse
behavior: worst in FC-12 than the lipid. This lipid is a very mild
solubilizing/reconstituting agent that has already been successfully
used for structure determination. Any advice or thoughts will be
greatly appreciated. Is this something that some of you have already
observed?
Many thanks in advance,
--
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab (310)-983-3516
email pe...@mednet.ucla.edu <mailto:pe...@mednet.ucla.edu>
--
Vincent Chaptal, PhD
Institut de Biologie et Chimie des Protéines
Drug-resistance modulation and mechanism Laboratory
7 passage du Vercors
69007 LYON
FRANCE
+33 4 37 65 29 01
http://www.ibcp.fr
