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Pascal,
Not personally observed this myself, but just wondering, as a control, have you tried setting trays with just the buffer+detergent (no PDC)? And if so, do
you see the same behavior or not? Also, do you see phase separation in all drops or just a majority? Do the drops that exhibit this phase separation cluster into certain precipitant groups or salts by any chance (ie, only low MW PEGs, only in MPD, etc)?
Cheers,
Nick
From: Pascal Egea [mailto:[log in to unmask]]
Sent: Thursday, August 01, 2013 6:24 PM
To: [log in to unmask]
Subject: [ccp4bb] membrane protein and phase separation
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 [log in to unmask]