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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