Re: [ccp4bb]: Imidazole in crysatllization

[andRe ambrosio]

Hi people, I completely agree with Tim' s opinion about this issue. However, when I' going to design a purification/crystallization protocol (I must say that I am NOT that experienced), I always choose to follow first the simplest way to go through it. I really like affinity columns (speacily the one that says single-step purification). In my case, eluting the protein from a Co2+ resin (~ 200mM imidazole), it comes out completely clean. To crystallize it i just cut the his-tag O/N, get the desired concentration in the next morning (keeping imidazole, clived histag and even the few thrombin) and set trays. In this case crystals (big and beautiful ones, in diffraction terms) pop out in less than 24 hours @ 18C. If I try to "improve" the protocol, removing all these "extra" agents, the solubilty of the sample changes completely and I still couldnt get crystals in any other conditions. Lucky with your experiments, -andre ambrosio Tim Gruene wrote: *** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk *** Hallo Nada, there are probably many people who made good experience with the presence of imidazole for crystallisation as there are many with bad experience. However, from what you were writing it sounds as though you want to crystallise the protein straight from the Ni-column. From my experience - and I dare say most people will agree - it is a good idea to have more than one or two purification steps. And a side effect of e.g. a size exclusion column would be that you can conveniently get rid of the imidazole, and most protein crystallographers I met would probably do so. If, however, your protein likes imidazole or even needs it for solubility, then obviously you keep the concentration throughout your purification. Hope this helps, Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Wed, 14 Sep 2005, Nada wrote: Hello. Please I would like to know if someone has had some experience with crystallization of protein in solution containing also 1M imidazole. Is it necessary to remove it from solution after elution of protein from Ni-column? Thank you Nada Pavlendova IMB, Bratislava, Slovakia