Pascal, The reason this phenomenon looks odd to you is that detergent phase separation is not a micelle size phenomenon, but a micelle surface phenomenon. Like any colloidal solution, even protein, there can be conditions where the colloidal particles aggregate (the cloud point), creating a mixture of solubilized colloidal particle L' and a second phase of aggregated particles L''. The second phase of aggregated particles (which can appear to be a viscous liquid) can be micelles, PDCs, or simply protein when working with only soluble proteins; for the latter two, this can lead to precipitation or crystals. However, at the concentrations of detergent/lipid we typically use, we tend to see only aggregates of micelles or PDCs in the second phase, rather than the other surfactant phases (bicubic, lamellar, hexagonal, etc.). Nor is the second phase formation an indication of micelle fusion (which was a hot topic of discussion in the 1980's).
The physical conditions that cause phase separation are the same as for crystallization (the solvent environment variables of pH, salt, polymer solutes, etc.). It can also be a unique phenomenon of the PDCs you make with your protein and the chosen detergent. One quick experiment you can try is to add glutaraldehyde to the reservoir and fix a drop with phase separation (glutaraldehyde is quite volitile). CAVEAT: you can't do this in the presence of Tris, free amine solutes like ammonium sulfate, or lipids/detergents with a free amine. If the droplets are fixed into a gel, then the predominant species in the second phase is not pure detergent micelles, but your PDCs at very high concentration (>50 mg/mL). Hence, your PDCs want to phase out, which is not always a bad thing. The trick then is to encourage crystallize. Good luck, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com **************************************************************** On Aug 1, 2013, at 6:23 PM, Pascal Egea wrote: > 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
