Dear Ivan, We faced the same problem for our Hya/Fab complex (clustered needles). By change the pH and doing microseeding, we were able to improve the crystals quality. For more information see our paper
Padavattan, S., Schirmer, T., Schmidt, M., Akdis, C., Valenta, R., Mittermann, I., Soldatova, L., Slater, J., Mueller, U., and Markovic-Housley, Z. (2007). Identification of a B-cell epitope of hyaluronidase, a major bee venom allergen, from its crystal structure in complex with a specific Fab. J Mol Biol 368, 742-752. for detailed protocol see the supplementary information in my thesis : http://edoc.unibas.ch/651/ We also faced the problem of antibody precipitation during dialysis and concentration with bacterial expressed IgG. This could be due to absence of glycosylation. So I avoided extensive dialysis and did papain digestion with diluted IgG. Digested sample load on to Mono-S column to separate the Fab isoforms from (Fab)2 and undigested IgG. The purified Fab mixed with antigen 1:1.2 molar ratio and used the gel filtration to separate the complex from unbound antigen. Hope this will help you Cheers Sivaraman Padavattan On Fri, Sep 10, 2010 at 8:29 AM, xaravich ivan <[email protected]>wrote: > 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 > >
