Adding DTT in your protein buffer or crystallization solution may also help. You could try increasing amounts of DTT/BME/TCEP in your crystallization solution and find a balance between reduction of skin formation vs getting crystals.
Adding 2mM DTT in my protein buffer helped me get rid of much of the skin on the drop in which the crystals were tightly embedded. -Gyan On Wed, Feb 25, 2015 at 3:00 AM, Han Remaut <han_c...@yahoo.co.uk> wrote: > Dear Ulrike, > > you could try avoid the drop-air interface by overlying sitting drops with > silicone oil or a 50/50 silicon/paraffin oil mixture. Note that this will > alter the kinetics with which your drops reach equilibrium, and hence may > alter your ability to get crystals of the protein. Batch crystallization > under oil is another option of course. > > Adding some alcohols (5-10% EtOH, isopropanol) or detergent (0.5 mM LDAO > for example) in your crystallization conditions may also be something to > consider. > > If none of these work, I'd concentrate on harvesting the crystals from the > skin. I tend to cut these open from the side, flip over the skin so that > one has better access to the crystals that generally are associated with > the inner face of the skin. You can try peel the crystals off the skin, or > cut out a piece of skin surrounding a crystal. That will not hurt > diffraction quality of the crystals. > > Hope that helps. > > Han > > > > On 25 Feb 2015, at 09:34, Ulrike Demmer wrote: > > > Dear crystallographers, > > > > I am trying to crystallize a soluble protein which tends to form > aggregates. The crystallization condition is 20% PEG 3350 + 0,2 M > Na-Formate. During the crstallization process a thick skin is formed on top > of the sitting-drops. As well the crystals are buried in precipitate. > Before I start harvesting I try to remove the skin but still it is hardly > possible to get any crystals out of these drops. > > > > Any suggestions how to avoid the formation of skin on crystallization > drops ? > > > > Cheers, > > > > Ulrike > > > Han Remaut, PhD > Laboratory of Structural & Molecular Microbiology > VIB / Vrije Universiteit Brussel > Building E4, Pleinlaan 2 > 1050 Brussel > > han.rem...@vib-vub.be > tel. +32-2-629 1923 / +32-499 708050 > http://www.vib.be/en/research/scientists/Pages/Han-Remaut-Lab.aspx -- Gyanendra Kumar, PhD St. Jude Children's Research Hospital, Department of Structural Biology, 262, Danny Thomas Place, MS-311 Memphis, TN 38105 Phone: 901-595-3839 Cell: 631-875-9189 -------------------------------------------------------
