Hi Adam, The diffraction improved from 8 Angstrom to 2.6 Angstrom. But, I had also removed the His-tag from the protein, so I am not sure how much was the role of this step and how much was it because of the addition of DTT. Two variables in one experiment :).
-Gyan On Wed, Feb 25, 2015 at 4:18 PM, Adam Brummett <[email protected]> wrote: > 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 <[email protected]> > 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 <[email protected]> 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 >> >> [email protected] >> 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 > ------------------------------------------------------- > > -- 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 -------------------------------------------------------
