See below. I believe Ray intended this to be sent to the entire bb and especially to Subbu!
And in reading Ray's message, I am reminded that I forgot to mention what he may be hinting at. You should also try to optimize by creating a fine grid screen around your identified condition varying in particular pH and precipitant concentration. best, Eric On Thu, 21 Jul 2011, ray-br...@att.net wrote: >What is the pI? Perhaps the protein will be more soluble close to this pH? >You could increase the salt and/or add some glycerol or a >cofactor? > >Normally crystallization is most affected by the pH, 2 - 10% glycerol or >temperature 4 - 25 degrees C. >Cheers > >Ray Brown > >_____________________________________________________________________________________________________________________________________ >From: Eric Larson <larso...@u.washington.edu> >To: CCP4BB@JISCMAIL.AC.UK >Sent: Thu, July 21, 2011 1:40:51 PM >Subject: Re: [ccp4bb] Concentrating a protein solution - subbu > >Hi Subbu, > >You got crystals at 1mg/ml so you probably don't need to concentrate your >protein any higher, especially since you suggest that >concentrating beyond that is problematic. Instead, you may want to try to >optimize the crystallization condition you have already >identified. Some possible things to try: additive screen, include specific >ligands, different temperature, different ratios of >protein solution to crystallization solution, seeding, different >crystallization methods (hanging vs. sitting drop, batch, >diffusion,...), ... > >good luck, > >Eric > >================================ >Eric T. Larson, PhD >Biomolecular Structure Center >Department of Biochemistry >Box 357742 >University of Washington >Seattle, WA 98195 > >email: larso...@u.washington.edu >================================ > >On Thu, 21 Jul 2011, Narayanan Ramasubbu wrote: > >> Dear All: >> We have been trying to crystallize a protein which is large - > 100 kDa. >> This is soluble but the best we can get is about 1 mg/mL. >> It did crystallize but did not diffract well. Efforts to increase the >> concentration has been unsuccessful. I am wondering whether >there are methods that others use to increase the concentration other that >using amicon columns. >> Any help will be appreciated. >> Thanks >> Subbu >> > >
