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 <e_rajaku...@yahoo.com>:
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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