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:ccp...@jiscmail.ac.uk] On Behalf Of Lari Lehtiö Sent: 18 February 2010 16:38 To: CCP4BB@JISCMAIL.AC.UK 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 <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) > > > Get your new Email address! > Grab the Email name you've always wanted before someone else does! > http://mail.promotions.yahoo.com/newdomains/aa/ > >
