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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