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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 <[log in to unmask]> 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 <[log in to unmask]> 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 <[log in to unmask]> 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

[log in to unmask]
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
-------------------------------------------------------