Thanks everyone, I have really a lot to do now. Jurgen, yes I do separate the complex over size-exclusion column before setting trays.
Patrick, Thanks for the wonderful reference. Sivaram, Thank you.It was really nice of you to send the link of your thesis.It is a wonderful gesture and I appreciate it. Could you remember how dilute your IgG starting sample was and the time of dialysis prior to papain digestion? Also could you elaborate a bit on how you specifically did the microseeding steps. Having a glance on your thesis, I could not find these of the top. Thanks again, Ivan On Fri, Sep 10, 2010 at 4:35 AM, Patrick Shaw Stewart <patr...@douglas.co.uk > wrote: > Again you should read the spectacular paper by Obmolova and co. where > they solved the structures of three Fab-antigen complexes using “MMS” > microseeding (seeding into random screens), starting with one hit containing > clusters of needles - which could not themselves be optimized > > > > The paper is available on open access (free) > > > > http://journals.iucr.org/d/issues/2010/08/00/issconts.html > > http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf > > > > *Acta Cryst.* (2010). D*66*, 927-933 [ > doi:10.1107/S0907444910026041<http://dx.doi.org/10.1107/S0907444910026041> > ] > Promoting crystallization of antibody-antigen complexes *via* microseed > matrix screening G. > Obmolova<http://scripts.iucr.org/cgi-bin/citedin?search_on=name&author_name=Obmolova%2C%20G%2E> > , T. J. > Malia<http://scripts.iucr.org/cgi-bin/citedin?search_on=name&author_name=Malia%2C%20T%2EJ%2E> > , A. > Teplyakov<http://scripts.iucr.org/cgi-bin/citedin?search_on=name&author_name=Teplyakov%2C%20A%2E> > , R. > Sweet<http://scripts.iucr.org/cgi-bin/citedin?search_on=name&author_name=Sweet%2C%20R%2E> > and G. L. > Gilliland<http://scripts.iucr.org/cgi-bin/citedin?search_on=name&author_name=Gilliland%2C%20G%2EL%2E> > > *Synopsis:** *The application of microseed matrix screening to the > crystallization of related antibodies in complex with IL-13 is described. > Both self-seeding or cross-seeding helped promote nucleation and increase > the hit rate. > > Online 14 July 2010 > > The application of microseed matrix screening to the crystallization > > of antibody–antigen complexes is described for a set > > of antibodies that include mouse anti-IL-13 antibody C836, its > > humanized version H2L6 and an affinity-matured variant of > > H2L6, M1295. The Fab fragments of these antibodies were > > crystallized in complex with the antigen human IL-13. The > > initial crystallization screening for each of the three complexes > > included 192 conditions. Only one hit was observed for H2L6 > > and none were observed for the other two complexes. Matrix > > self-microseeding using these microcrystals yielded multiple > > hits under various conditions that were further optimized to > > grow diffraction-quality H2L6 crystals. The same H2L6 seeds > > were also successfully used to promote crystallization of the > > other two complexes. The M1295 crystals appeared to be > > isomorphous to those of H2L6, whereas the C836 crystals were > > in a different crystal form. These results are consistent with the > > concept that the conditions that are best for crystal growth > > may be different from those that favor nucleation. Microseed > > matrix screening using either a self-seeding or cross-seeding > > approach proved to be a fast, robust and reliable method not > > only for the refinement of crystallization conditions but also to > > promote crystal nucleation and increase the hit rate. > > > > > > > > -- > > For information and discussion about protein crystallization and > automation, please join > > our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en > > > > patr...@douglas.co.uk Douglas Instruments Ltd. > > DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK > > Directors: Peter Baldock, Patrick Shaw Stewart > > http://www.douglas.co.uk/ > > Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 > > Regd. England 2177994, VAT Reg. GB 480 7371 36 > > > > *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of > *xaravich > ivan > *Sent:* 10 September 2010 04:00 > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] Fab purification and crystallization > > > > Hi CCP4bb, > > I have two questions regarding Fab purification and Fab-antigen complex > crystallization and would really appreciate any input from the experienced > board. > > 1) I have got some hits for Fab-antigen complex (150 kD) but they are all > needle clusters. Whatever fine screen I formulate, it always gives me these > needle clusters. Are there some better common ways to change needles to > single crystals? > > 2) I have certain IgGs from which I purify the Fab by papain digestion > (resin from ThermoSci). One of the first steps is to dialyze the IgG with > the digestion buffer,( 20mM Na-phosphate and 10mM EDTA pH 7.0) Over night. I > always get 30-60% of the IgG precipitated during this overnight dialysis. I > tried to increase the salt by adding 200mM NaCl but of no effect. Have > anyone experienced such problem? Is there any thing that could be tried to > stop this precipitation. > > thanks in advance. > > ivan >
