I see some solutions to your problem and one works well for me on a small protein domain.

I had exactly the same problem of crystallization during concentration and in my case I solve the problem by heating the crystal suspension. The validation of the protocol was made with support of 1D NMR to verify the stability of the structure after heating.

So the solutions I see are:
- Concentrate your sample until apparition of crystals and mildly heat it until solubilization of your crystals. Make a drop of 5µL in a crystallization plate and let it cool down against 500µL of your buffer.
- Concentrate your sample until apparition of crystals and add the necessary amount of water to solubilize your crystals. Make drops of 1 or 2µL in a crystallization plate and let it concentrate against 500µL of your buffer.
- You can also choose the best solution (heating or dilution) to solubilize your crystals ans try mild evaporation under paraffin oil to try to obtain crystals.

And after, with the best solution, you can play with the salt concentration of reservoir solution to try to obtain better crystals.

Gook luck.

Michel.

2010/2/18 Daniel Ryan <[log in to unmask]>

A cheaper solution than buying the dialysis buttons would be just to make your own out of the top of a microtube. Depending on what volume you want to dialyse you can use either a PCR tube (~35 uL per lid) or larger 1.5 mL tube (~250 uL). Just cut the top off the tube using a hot scalpel, you then place your sample in the cap of the microtube, cover it with dialysis membrane and then secure the tubing using the top part of the tube that you cut-off.

-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Lari Lehtiö
Sent: 18 February 2010 16:38
To: [log in to unmask]
Subject: Re: [ccp4bb] Protein crystallizes while concentration

Dear Rajkumar,

I would use dialysis buttons.
E.g. http://hamptonresearch.com/product_detail.aspx?cid=10&sid=63&pid=111

Put your protein to the button and seal it with a piece of dialysis
membrane. Place this to a linbro plate (easy to look with a
microscope), fill the well with low salt buffer and seal with a cover
slip.

To make the diffusion slower, you can put the dialysis button inside a
dialysis tubing with some high salt buffer and place this to a bigger
volume of low salt buffer.


~L~

______________________________________________________
Lari Lehtiö
Pharmacy, Department of Biochemistry and Pharmacy
Åbo Akademi University,
BioCity, FIN-20520 Turku
Finland
+358 2 215 4270
http://www.users.abo.fi/llehtio/
______________________________________________________


Quoting E rajakumar <[log in to unmask]>:

> Dear All
> I am Rajkumar, working on the protein which has unusual behavior
> while concentration.
>
> When I kept protein in 15 mM Tris 7.5, 150 mM NaCl and 5 mM DTT, the
>  solubility of the protein is decreases drastically and tend to
> crystallize while concentration.
> Protein cannot be concentrated more than 3 mg/mL, however I noticed
> white turbid protein if I force to concentrate >3mg/mL. When I
> observed this white turbid solution under the microscope, I noticed
> shower of tiny protein crystals which are needle in shape.
> I screened freshly purified protein (2.5 mg/mL) in different Hampton
>  and Qiagen screens, strangely none of the conditions gave the
> crystals.  I concentrated left over protein at 15oC at 3 mg/mL and
> kept in the 4oC for 4 days again I noticed shower of crystals.
> This protein solubility is increased to ~20mg/mL when I kept in 15
> Tris 7.5, 400 mM NaCl, 5% Glycerol and 5 mM DTT, but did not
> crystallize while concentration and also after screening with
> Hampton and Qiagen screens.
>
> My queries are
> 1. How do I get the crystals in the crystallization set up rather
> than while concentration, so that I can control the diffusion and
> finally nucleation?
> 2. Could anybody give me suggestions on seeding in this type of situation?
> 3. Any comments on reverse vapor diffusion for this type of protein
> are most welcome. So I can keep protein in high ionic strength (~400
>  mM NaCl)and diffuse against low Ionic strength or deionized water?
> Or any other protocol?
> Any suggestions are well appreciated.
> Thanking you in advance
> Raj
>
> E. Rajakumara
> Postdoctoral Fellow  Strcutural Biology Program
> Memorial Sloan-Kettering Cancer Center
> New York-10021
> 001 212 639 7986 (Lab)  001 917 674 6266 (Mobile)
>
>
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