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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) > > > Get your new Email address! > Grab the Email name you've always wanted before someone else does! > http://mail.promotions.yahoo.com/newdomains/aa/ > >