Re: [ccp4bb] isopropanol as a precipitant
Shivesh It's often very difficult to harvest crystals from conditions that contain isopropanol because the alcohol is volatile - especially with 50% or over! A solution that has worked several times it so use the Vapor Batch plates. http://www.douglas.co.uk/vb.htm You can cover the drops with oil, and then put isopropanol solution into the moat of the plate. The alcohol diffuses through the oil into the drops, while the oil acts as a buffer, reducing loss of alcohol during harvesting and freezing. For a case study see http://www.douglas.co.uk/winner1.htm Please let me know if anyone wants samples. Patrick -- [EMAIL PROTECTED]Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart, James Smith http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of shivesh kumar Sent: 05 March 2008 07:23 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] isopropanol as a precipitant Dear all Sorry for non-CCP4 query. I have crystallized a 7kDa protein in 50-60% of isopropanol,pH 4.0-4.6.The interesting thing is that the xtal appears within 5 hrs at 16 degree. The protein conc is 5mg/ml and drop size is 3+1 with mother liquor 300 micro lt.Numerous xtals appears and but small is size.Do anyone can share their experience with Isopropanol as a precipitant and to improve the xtal quality with other precipitants.All suggestions are welcome. Thanx in advance. Shivesh
Re: [ccp4bb] isopropanol as a precipitant
Hi, if you look at papers on crystallization of the E.coli protein ROP, you might find interesting info, in particular e.g.: Papanikolau Y, Kotsifaki D, Fadouloglou VE, Gazi AD, Glykos NM, Cesareni G, Kokkinidis M. Ionic strength reducers: an efficient approach to protein purification and crystallization. Application to two Rop variants. Acta Crystallogr D Biol Crystallogr. 2004 Jul;60(Pt 7):1334-7 (sounds like a lot of work, but you asked for it ... ;-) ) volatility is obviously a big problem, but as for cryo use paratone-N (or related dry highly viscous, see previous discussions on its behaviour on ccp4bb) oil, thats probably the easiest thing to do. (only thing ever work for me with methanol and ethanol) obvioulsy you can try butanols etc and MDP as the less volatile relatives. HTH, Tommi ps sounds suspiciously close to ROP in size... not a fourhelix bundle is it??? (none of my business obviously though) (on another note i think it would be nice if people would subscribe with their institute email addresses so that we are all on the same level in public discussions. not that it should be mandatory but it would be sort of nice given its a professional forum) Quoting shivesh kumar [EMAIL PROTECTED]: Dear all Sorry for non-CCP4 query. I have crystallized a 7kDa protein in 50-60% of isopropanol,pH 4.0-4.6.Theinteresting thing is that the xtal appears within 5 hrs at 16 degree. The protein conc is 5mg/ml and drop size is 3+1 with mother liquor 300 micro lt.Numerous xtals appears and but small is size.Do anyone can share their experience with Isopropanol as a precipitant and to improve the xtal quality with other precipitants.All suggestions are welcome. Thanx in advance. Shivesh -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
[ccp4bb] isopropanol as a precipitant
Dear all Sorry for non-CCP4 query. I have crystallized a 7kDa protein in 50-60% of isopropanol,pH 4.0-4.6.Theinteresting thing is that the xtal appears within 5 hrs at 16 degree. The protein conc is 5mg/ml and drop size is 3+1 with mother liquor 300 micro lt.Numerous xtals appears and but small is size.Do anyone can share their experience with Isopropanol as a precipitant and to improve the xtal quality with other precipitants.All suggestions are welcome. Thanx in advance. Shivesh
