Gyan, With the addition of DTT to remove the skin, did you see an increase in the resolution with the skin no longer present compared to with it still there?
-Adam > On Feb 25, 2015, at 11:15 AM, Gyanendra Kumar <gyanendr...@gmail.com> wrote: > > 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 > -------------------------------------------------------
